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PasSenger Mutations Confound Interpretation Of All Genetically Modified Congenic Mice
Abstract
Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived)
and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.
... Genetically modified mice are usually generated using germline transmission competent embryonic stem (ES) cell lines derived from the 129 mouse strain (15,16). 129 ES cell lines are easy to handle in culture and remain competent to repopulate the mouse germline. ...
... For phenotypic studies, 129 chimeric mice are backcrossed repeatedly to a particular strain, often C57BL/6, whereby the genomic signature of the congenic mice (129/B6) depends on the number of backcrosses. Backcrossing for more than 10 generations is desirable to increase the genetic homogeneity (18) but to decrease the impact of passenger mutations that affect phenotypes (16), genetic matters critical for the validation and reproducibility of experimental studies (19). ...
... 7-10 generations) of genetically modified mice with inbred mouse strains such as the C57BL/6 substrains B6N or B6J, as well as the breeding strategies across generations might influence the phenotypic outcomes (49). For example, B6J mice are more susceptible to lipopolysaccharideand tumor necrosis factor a-induced lethal shock as compared to B6N/B6J mice (16). In tumor development, the lymphoma formation in Pten-deficient mice depends on C57BL/6 background, but not Balb/c background (50). ...
Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1-/-, Cybb-/- or Casp1-/-/Cybb-/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.
... In the mouse, 1 cM is, on average, equivalent to 2,000 kilobases; however, the rate of equivalence can vary greatly due to numerous factors (Silver, 1995). This implicates a probability of about 90% that upon backcrossing for 10 generations; a flanking region with a distance of 1 cM to the targeted gene is still of donor origin (Berghe et al., 2015;Lusis et al., 2007). Gene expression can vary in cells from different laboratory mouse strains and some mouse strains even constitute ''natural knockouts'' for certain genes e.g. if mutations or SNPs introduce premature STOP codons . ...
... The 129 mouse strains carry a defective Casp11 gene in the close proximity of Casp1, and the strong resistance to lethal lipopolysaccharide (LPS) injection found in Casp1 À/À mice was mainly due to the defective Casp11 neighboring gene that was carried along as passenger mutation (Kayagaki et al., 2011). A later study could show that targeting of other Casp11 neighboring genes, such as Panx1, also led to a resistance toward lethal LPS injection, if ESCs were derived from 129 mice, but not when B6-derived ESCs were used (Berghe et al., 2015). These two examples illustrate the potential impact of 129-derived passenger mutations and possible consequences, if they remain undetected. ...
... The role of the inactive Casp11 passenger gene in LPS resistance was unnoticed for more than 15 years until Kayagaki and colleagues revealed this important fact (Kayagaki et al., 2011). The defective Casp11 gene was also found in other transgenic mice generated with 129-derived ES cells, such as Panx1 tm1.1Vshe (Dvoriantchikova et al., 2012), again conferring these transgenic mice resistance to LPS (Berghe et al., 2015). These examples illustrate the importance of screening transgenic mice with a congenic background for passenger mutations in order to avoid experimental bias. ...
Among laboratory mouse strains many genes are differentially expressed in the same cell population. As consequence, gene targeting in 129-derived embryonic stem cells (ESCs) and backcrossing the modified mice onto the C57BL/6 background can introduce passenger mutations in the close proximity of the targeted gene. Here, we demonstrate that several transgenic mice carry a P2rx7 passenger mutation that affects the function of T cells. By the example of P2rx4tm1Rass we demonstrate that P2X4ko T cells express higher levels of P2X7 and are more sensitive toward the P2X7 activators ATP and NAD+, rendering these cells more vulnerable toward NAD-induced cell death (NICD) compared with wild type (WT). The enhanced NICD sensitivity confounded functional assays e.g. cytokine production and cell migration. Our results need to be considered when working with P2rx4tm1Rass mice or other 129-based transgenic strains that target P2rx7 neighboring genes.
... In the current study we show, using in silico modelling, how these substitutions affect the interaction with BACE1. We have also used CRISPR-Cas9 technology to scarlessly humanize the endogenous Aβ sequence in the mouse and rat App genes (15) and investigate the effects on APP processing. We also created a PSEN1 knock-in mutation to generate a rat model for AD. ...
... B63 antibody detects full length APP (APP FL) and C-terminal fragments (CTF, longer exposure). 82E1 antibody speci cally detects human AB (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), and hence the human B-CTF, independently con rming that the mouse App gene was successfully humanized. β-ACTIN was used as loading control. ...
... B63 antibody binds to full length APP (FL APP) and C-terminal fragments (CTF, longer exposure). 82E1 antibody speci cally detects human Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), and hence the human B-CTF, independently con rming that the rat App gene was successfully humanized. β-ACTIN was used as a loading control. ...
Background
Three amino acid differences between rodent and human APP affect medically important features, including β-secretase cleavage of APP and Aβ peptide aggregation(1–3). Most rodent models for Alzheimer’s disease (AD) are, therefore, based on the human APP sequence, expressed from artificial mini-genes randomly inserted in the rodent genome. While these models mimic rather well various biochemical aspects of the disease, such as Aβ-aggregation, they are also prone to overexpression artifacts and to complex phenotypical alterations, due to genes affected in or close to the insertion site(s) of the mini-genes(4,5). Knock-in strategies which introduce clinical mutants in a humanized endogenous rodent APP sequence(6) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice. Methods
Computational modelling of the human β-CTF bound to BACE1 was used to study the differential processing of rodent and human APP. We humanized the three pivotal residues we identified G676R, F681Y and R684H (labeled according to the human APP770 isoform) in the mouse and rat genomes using a CRISPR-Cas9 approach. These new models, termed mouse and rat App hu/hu , express APP from the endogenous promotor. We also introduced the early-onset familial Alzheimer’s disease (FAD) mutation M139T into the endogenous Rat Psen 1 gene.ResultsWe show that introducing these three amino acid substitutions into the rodent sequence lowers the affinity of the APP substrate for BACE1 cleavage. The effect on β-secretase processing was confirmed as both humanized rodent models produce three times more (human) Aβ compared to the original WT strain. These models represent suitable controls, or starting points, for studying the effect of transgenes or knock-in mutations on APP processing(6). We introduced the early-onset familial Alzheimer’s disease (FAD) mutation M139T into the endogenous Rat Psen 1 gene and provide an initial characterization of Aβ processing in this novel rat AD model.Conclusion The different humanized APP models (rat and mouse) expressing human Aβ and PSEN1 M139T are valuable controls to study APP processing in vivo allowing the use of a human Aβ ELISA which is more sensitive than the equivalent system for rodents. These animals will be made available to the research community.
... Alternatively, discrepancies between our results and others may be related to differences in phenotype caused by mouse genetic background (Gerlai, 1996;Simpson et al., 1997) and/or effects of passenger mutations in Panx1 congenic mice (Vanden Berghe et al., 2015). An example from a previous study found that two Panx1-null mouse lines (Panx1 Vsh and Panx1 Vmd , derived, respectively, from 129 and C57Bl/6 embryonic stem cells) expressed the presence or absence of a phenotype that coincided with the presence or absence of one passenger mutation (Vanden Berghe et al., 2015). ...
... Alternatively, discrepancies between our results and others may be related to differences in phenotype caused by mouse genetic background (Gerlai, 1996;Simpson et al., 1997) and/or effects of passenger mutations in Panx1 congenic mice (Vanden Berghe et al., 2015). An example from a previous study found that two Panx1-null mouse lines (Panx1 Vsh and Panx1 Vmd , derived, respectively, from 129 and C57Bl/6 embryonic stem cells) expressed the presence or absence of a phenotype that coincided with the presence or absence of one passenger mutation (Vanden Berghe et al., 2015). Thus, it is possible that the potentiation of LTP and absence of LTD found in the two 129-derived Panx1 mouse lines (Panx1 −/− and Panx1 Vsh (Ardiles et al., 2014;Prochnow et al., 2012) and the attenuation of LTP and LTD we observed for the Panx1 KOMP (C57BL/6-derived ECS)) could be due to a passenger mutation. ...
Pannexin 1 (Panx1) is an ubiquitously expressed protein that forms plasma membrane channels permeable to anions and moderate-sized signaling molecules (e.g., ATP, glutamate). In the nervous system, activation of Panx1 channels has been extensively shown to contribute to distinct neurological disorders (epilepsy, chronic pain, migraine, neuroAIDS, etc.), but knowledge of the extent to which these channels have a physiological role remains restricted to three studies supporting their involvement in hippocampus dependent learning. Given that Panx1 channels may provide an important mechanism for activity-dependent neuron-glia interaction, we used Panx1 transgenic mice with global and cell-type specific deletions of Panx1 to interrogate their participation in working and reference memory. Using the eight-arm radial maze, we show that long-term spatial reference memory, but not spatial working memory, is deficient in Panx1-null mice and that both astrocyte and neuronal Panx1 contribute to the consolidation of long-term spatial memory. Field potential recordings in hippocampal slices of Panx1-null mice revealed an attenuation of both long-term potentiation (LTP) of synaptic strength and long-term depression (LTD) at Schaffer collateral-CA1 synapses without alterations of basal synaptic transmission or pre-synaptic paired-pulse facilitation. Our results implicate both neuronal and astrocyte Panx1 channels as critical players for the development and maintenance of long-term spatial reference memory in mice.
... Specifically, the interpretation of results of genetic and pharmacological experiments presented herein should place particular attention on the aforementioned connectivity amongst different RCD variants as well as on discriminating between essential vs. accessory aspects of cell death [14]. Another issue to be considered is the fact that most conclusions are based on use of knockout/congenic mice which often present other passenger mutations potentially influencing the observed phenotype [73]. Our objective is not only to provide a critical summary of the existing literature, but also to offer an updated framework for interpretation of these findings in view of currently accepted models of RCD signaling. ...
... BIM, BID and BAD have all been shown to influence survival in mouse models of septic shock, as their targeting confer protective effects from tissue damage of multiple organs [439][440][441], as well as in patients with severe sepsis [442]. On the contrary, the role of apoptotic caspases in septic shock is contentious [54,73,443,444]. The precise impact of apoptosis in widespread inflammation during sepsis requires further investigation. ...
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
... Alternatively, discrepancies between our results and others may be related to differences in phenotype caused by mouse genetic background (Gerlai 1996;Simpson et al., 1997) and/or effects of passenger mutations in Panx1 congenic mice (Vanden Berghe et al., 2015). An example from a previous study found that two Panx1-null mouse lines (Panx1 Vsh and Panx1 Vmd , derived respectively from 129 and C57Bl/6 embryonic stem cells) expressed the presence or absence of a phenotype that coincided with the presence or absence of one passenger mutation (Vanden Berghe et al., 2015). ...
... Alternatively, discrepancies between our results and others may be related to differences in phenotype caused by mouse genetic background (Gerlai 1996;Simpson et al., 1997) and/or effects of passenger mutations in Panx1 congenic mice (Vanden Berghe et al., 2015). An example from a previous study found that two Panx1-null mouse lines (Panx1 Vsh and Panx1 Vmd , derived respectively from 129 and C57Bl/6 embryonic stem cells) expressed the presence or absence of a phenotype that coincided with the presence or absence of one passenger mutation (Vanden Berghe et al., 2015). Thus, it is possible that the potentiation of LTP and absence of LTD found in the two 129-derived Panx1 mouse lines (Panx1 -/and Panx1 Vsh : (Ardiles et al., 2014;Prochnow et al., 2012) and the attenuation of LTP and LTD we observed for the Panx1 KOMP (C57BL/6-derived ECS)) could be due to the passenger mutation. ...
Pannexin 1 (Panx1) are ubiquitously expressed proteins that form plasma membrane channels permeable to anions and moderate sized signaling molecules (e.g., ATP, glutamate). In the nervous system, activation of Panx1 channels have been extensively shown to contribute to distinct neurological disorders (epilepsy, chronic pain, migraine, neuroAIDS, etc.) but knowledge of extent to which these channels have a physiological role remains restricted to three studies supporting their involvement in hippocampus dependent learning. Given that Panx1 channels may provide an important mechanism for activity-dependent neuron glia interaction, we used Panx1 transgenic mice with global and cell-type specific deletions of Panx1 to interrogate their participation in working and reference memory. Using the 8-arm radial maze, we show that long-term spatial reference memory, but not spatial working memory, is deficient in Panx1-null mice and that both astrocyte and neuronal Panx1 contribute to the consolidation of long-term spatial memory. Field potential recordings in hippocampal slices of Panx1 null mice revealed an attenuation of both long-term potentiation (LTP) of synaptic strength and long-term depression (LTD) at Schaffer collateral CA1 synapses without alterations basal synaptic transmission or pre-synaptic paired-pulse facilitation. Our results implicate both neuronal and astrocyte Panx1 channels as critical players for the development and maintenance of long-term spatial reference memory in mice.
... To this end, we provide a framework for neuroscientists of varying expertise and area of research focus rooted in openscience principles and user-friendly resources. We highlight the standard steps and consideration of a research study investigating cell-types in mouse brain, including: statistical power and sample size estimation (Button et al., 2013;Picciotto, 2020), selection of control groups (Tye, 2018;discussed in Picciotto, 2019), sex and genetic background (Kiselycznyk and Holmes, 2011;Vanden Berghe et al., 2015;McCarthy et al., 2017;Luo et al., 2020), image acquisition and processing Heddleston et al., 2021), and data visualization and statistical analysis (Calin-Jageman and Cumming, 2019) (Figure 1). Moreover, we provide a step-by-step FIJI-ImageJ image analysis workflow to quantify cell density in the mouse brain sections. ...
... We recommend that researchers refer to the Mouse Genome Informatics website 3 3 http://www.informatics.jax.org/ for precise information (e.g., nomenclature, strain-specific single nucleotide polymorphism, phenotypes, gene expression, and Cre-recombinase activity), especially "Me-PaMuFind-It" 4 to identify potential 129S-derived mouse strain passenger mutations (particularly if the strain has not been backcrossed substantially). Consistent use of these resources is critical to prevent and mitigate possible confounds secondary to germline recombination (Luo et al., 2020) and passenger mutations (Vanden Berghe et al., 2015). ...
The ever-expanding availability and evolution of microscopy tools has enabled ground-breaking discoveries in neurobiology, particularly with respect to the analysis of cell-type density and distribution. Widespread implementation of many of the elegant image processing tools available continues to be impeded by the lack of complete workflows that span from experimental design, labeling techniques, and analysis workflows, to statistical methods and data presentation. Additionally, it is important to consider open science principles (e.g., open-source software and tools, user-friendliness, simplicity, and accessibility). In the present methodological article, we provide a compendium of resources and a FIJI-ImageJ-based workflow aimed at improving the quantification of cell density in mouse brain samples using semi-automated open-science-based methods. Our proposed framework spans from principles and best practices of experimental design, histological and immunofluorescence staining, and microscopy imaging to recommendations for statistical analysis and data presentation. To validate our approach, we quantified neuronal density in the mouse barrel cortex using antibodies against pan-neuronal and interneuron markers. This framework is intended to be simple and yet flexible, such that it can be adapted to suit distinct project needs. The guidelines, tips, and proposed methodology outlined here, will support researchers of wide-ranging experience levels and areas of focus in neuroscience research.
... At the same time, only mild phenotypes were observed in Pannexin1 knockout mice [20]. In addition, it was reported that a passenger mutation in the Casp11 gene was identified in a popular line of pannexin 1 knockout mice, questioning even those effects [31]. Here we reduplicated pannexin 1 knockout mice using the CRISPR/Cas9 method and reproduced substitution at position 217 (p.Arg217His) in the mice model. ...
... Although numerous backcrossing strategy is used to omit ESC-derived genetic background effects of this approach, genetically engineered animals can retain passenger mutations in the region closely flanking the targeted gene. Comparative genomics analysis between C57BL/6 and the 129 strains [31] identified multiple passenger mutations (1084 genes are predicted to be aberrantly expressed). In the popular Panx1 knockout strain [34] Casp11, an inactivating passenger mutation was found that can produce "false-positive" phenotypes. ...
In humans and other vertebrates pannexin protein family was discovered by homology to invertebrate gap junction proteins. Several biological functions were attributed to three vertebrate pannexins members. Six clinically significant independent variants of the PANX1 gene lead to human infertility and oocyte development defects, and the Arg217His variant was associated with pronounced symptoms of primary ovarian failure, severe intellectual disability, sensorineural hearing loss, and kyphosis. At the same time, only mild phenotypes were observed in Panx1 knockout mice. In addition, a passenger mutation was identified in a popular line of Panx1 knockout mice, questioning even those effects. Using CRISPR/Cas9, we created a new line of Panx1 knockout mice and a new line of mice with the clinically significant Panx1 substitution (Arg217His). In both cases, we observed no significant changes in mouse size, weight, or fertility. In addition, we attempted to reproduce a previous study on sleep/wake and locomotor activity functions in Panx1 knockout mice and found that previously reported effects were probably not caused by the Panx1 knockout itself. We consider that the pathological role of Arg217His substitution in Panx1, and some Panx1 functions in general calls for a re-evaluation.
... Assessing Gba KI and WT mice for mutations linked to retinal pathology. Since mutations for Rd1, Rd8, Rd10 and Rd12, have been linked to retinal pathology in mice, we genotyped our mice for these mutations to assess if the phenotypes in our study could possibly be due to any of these mutations acting as passenger mutation of the Gba knock-in [28][29][30][31] . All mice were wild-types for Rd1, Rd10 and Rd12. ...
... Indeed, all the Gba WT and KI mice used for the data analyses of the main paper were homozygous for the Rd8 mutation. This shows that the Rd8 mutation is not a passenger mutation of the Gba knock-in [28][29][30][31] . Yet, while these experimental groups were identical for the Rd8 mutation, and we have not detected overt retinal lesions of the type described by 25 , we cannot exclude that an interaction of the Rd8 mutation with the Gba KI and possibly age contributes to the retinal thinning reported here. ...
Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson’s Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1D409V/D409V knock-in (Gba KI/KI; “KI”) mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.
... Differently from C57BI/6J [57], we do not observe biologically significant levels of necroptotic cell death in the liver which was shown to be the main driver of TNF-induced mortality in this background strain. It was previously shown that C57BL/6J are exquisitely sensitive to TNF-induced shock than C57BL/6N due to presence of several passenger mutations in C57BI/6N [74], suggesting that these different backgrounds differentially influence the organ susceptibility of the mice to TNF-induced shock. ...
Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl −/− or Ripk3 −/− mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.
... From Table 1 it is obvious that an evenness in the use of littermate controls and comparison between appropriate genetic backgrounds in the experimental setup is also essential for reliable conclusions between genetically modified mouse strains [9,38]. ...
The involvement of necroptosis in the control of influenza A virus (IAV) infection has been reported in multiple studies. Downstream of the nucleic acid sensor ZBP1, RIPK3 kinase activity is critically involved in the induction of necroptotic cell death by phosphorylating MLKL, while RIPK3 as a scaffold can induce apoptosis. Paradoxically, RIPK3 -deficiency of mice may result in increased or decreased susceptibility to IAV infection. Here, we critically review the published reports on the involvement of RIPK3 in IAV infection susceptibility and try to identify differences in experimental settings that could explain seemingly conflicting outcomes. Analysis of the experimental reports revealed differences in the IAV challenge dose, the IAV inoculum preparation, IAV titer assessment, as well as the route of inoculation between studies. Furthermore, differences were noticed in the inclusion of littermate controls, which show high variance in viral sensitivity. Our evaluation argues for a standardized setup for IAV infection experiments including the preparation of the IAV virus, the use of different IAV infectious doses description and the proper experimental genetic controls of the mouse strains to increase inter-laboratory consistency in this field.
... However, such conclusions must be reinterpreted under the light of new experiments because they were originally performed on a knockout strain that was also deficient in caspase-11 [42,114]. Indeed, the same caspase-11 inactivating mutation exists in many mouse models, clouding the interpretation of the role of proposed inflammatory mediators based on the 129 mouse genetic background [115]. For example, this mutation misguided the interpretation of the caspase-11 role in septic shock models [42]. ...
The protein scaffold that includes the caspases is ancient and found in all domains of life. However, the stringent specificity that defines the caspase biologic function is relatively recent and found only in multicellular animals. During the radiation of the Chordata, members of the caspase family adopted roles in immunity, events coinciding with the development of substrates that define the modern innate immune response. This review focuses on the switch from the non-inflammatory cellular demise of apoptosis to the highly inflammatory innate response driven by distinct members of the caspase family, and the interplay between these two regulated cell death pathways.
... There are several limitations to this study that should be considered. First, while the Slpi −/− mice used in our study were backcrossed 10+ generations, we cannot exclude the possibility that passenger mutations (90) affecting genes in regions flanking Slpi (e.g. Serinc3 and Pkig) indirectly influence our phenotypes. ...
Annually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.
... While remarkable progress has been made in this area, there are still only a few drugs that can inhibit caspase-11 non-canonical inflammasome. Despite the fact that several of the caspases-4 small molecule inhibitors and GSDMD pore-formation inhibitors recently identified in studies have received FDA medication approval [7,51,52], they share many of the same negative effects as nonsteroidal anti-inflammatory drugs. It is most noteworthy that in the absence of caspase-11, mice develop an acute susceptibility to infection from bacteria such Burkholderia pseudomallei and B. thailandensis that reproduce in the cytosol after escaping the phagosome [53]. ...
Septic shock is defined as a subset of sepsis, which is associated with a considerably high mortality risk. The caspase-11 non-canonical inflammasome is sensed and activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis, it plays a critical role in septic shock. However, there are few known drugs that can control caspase-11 non-canonical inflammasome activation. We report here that goitrin, an alkaloid from Radix Isatidis, shows protective effects in LPS-induced septic shock and significant inhibitory effect in caspase-11 non-canonical inflammasome pathway. Male C57BL/6J were injected intraperitoneally with LPS (20 mg/kg) to induce experimental septic shock. The results demonstrated that the survival rates of mice pretreated with goitrin or Toll-like receptor 4 (TLR4) inhibitor TKA-242 increased, and LPS-induced hypothermia and lung damage improved by inhibiting inflammatory response. Elucidating the detailed mechanism, we surprisingly found goitrin is really different from TAK-242, it independent of the TLR4 signal activation, but significantly inhibited the activation of caspase-11 non-canonical inflammasome, including cleaved caspase-11 and N-terminal fragment of gasdermin D (GSDMD-NT). Furthermore, with a nonlethal dose of the TLR3 agonist poly(I:C)-primed and subsequently challenged with LPS to induce caspase-11-mediated lethal septic shock, the efficacy of goitrin had been verified. Those results revealed the effect of goitrin in protective against LPS-induced septic shock via inhibiting caspase-11 non-canonical inflammasome, which provided a new therapeutic strategy for clinical treatment of septic shock.
... Additionally, the most commonly used Mlkl -/mouse lines were generated and are maintained on a C57BL/6 J background [4,12], while the most used Ripk3 -/mouse line is C57BL/6NJ [14]. Despite being derived from the same original mouse strain, 6 J and 6NJ are separated by over 60 years of independent breeding and demonstrate profound differences in morbidity and mortality following inflammatory challenges, for example, in LPS-or TNFαinduced sepsis [31] and Influenza A virus infection [32]. ...
MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl –/– and Ripk3 –/– mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl –/– female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.
... In another example, caspase-3 KO C57BL/6 mice produced using 129 ESC were also resistant to septic shock mortality due to the strain 129 caspase-11 passenger gene (Vanden Berghe et al., 2013). There are over a thousand 129 strain genes, which diverged from the C57BL/6 strain sequence (Vanden Berghe et al., 2015) and 12 backcrosses over 2 years of breeding have been estimated to result in C57BL/6 mice with 1% 129 genes or about 300 129 genes (Festing, 1992). However, by now most newly generated KO strains are based on targeting ES cells derived from C57BL/6 mice, allowing for defined background genetics in the animal studies (Pettitt et al., 2009). ...
Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.
... The difference could, however, also be due to "passenger genes" from the 129 embryonic stem cell donor that are transferred along with the gene of interest. A number of methods have been proposed for separating the gene of interest from the flanking region to eliminate the passenger genes (91)(92)(93). ...
Genetically modified mice are engineered as models for human diseases. These mouse models include inbred strains, mutants, gene knockouts, gene knockins, and ‘humanized’ mice. Each mouse model is engineered to mimic a specific disease based on a theory of the genetic basis of that disease. For example, to test the amyloid theory of Alzheimer’s disease, mice with amyloid precursor protein genes are engineered, and to test the tau theory, mice with tau genes are engineered. This paper discusses the importance of mouse models in basic research, drug discovery, and translational research, and examines the question of how to define the “best” mouse model of a disease. The critiques of animal models and the caveats in translating the results from animal models to the treatment of human disease are discussed. Since many diseases are heritable, multigenic, age-related and experience-dependent, resulting from multiple gene-gene and gene-environment interactions, it will be essential to develop mouse models that reflect these genetic, epigenetic and environmental factors from a developmental perspective. Such models would provide further insight into disease emergence, progression and the ability to model two-hit and multi-hit theories of disease. The summary examines the biotechnology for creating genetically modified mice which reflect these factors and how they might be used to discover new treatments for complex human diseases such as cancers, neurodevelopmental and neurodegenerative diseases.
... As per classical transgenic mice technology, the MRPS5 mutations were introduced into embryonic stem (ES) cells of Sv129 genetic background and then transferred to a C57BL/6 background by successive backcrossing. Inherently, the genomic region flanking the targeted gene in mutant mice (but not in the WT littermate control) was carried from the Sv129 background during backcrossing and might affect the phenotypic outcome [30]. RNA-seq data revealed that the size of the Sv129-derived donor region in MRPS5 V338Y/V338Y and MRPS5 G315R/G315R mice was similar, with approximately 25 Mb and 50 Mb, respectively. ...
We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.
... C57BL/6J Mlkl −/− mice were a gift from Dr. Warren Alexander and Dr. James Murphy (Walter & Eliza Hall Institute of Medical Research) [63]. Ripk3 −/− mice originally in C57BL/6N background were a gift from Dr. Vishva Dixit (Genentech, San Francisco) [64], but further backcrossed into C57BL/6J background for more than 5 generations in the laboratory, as all other strains were C57BL/6J and we reported earlier marked difference between C57BL/6J and C57BL/6N in various animal models [65]. All mice were maintained by heterozygote breeding to produce littermate controls for the experiments. ...
Cancers acquire several capabilities to survive the multistep process in carcinogenesis. Resisting cell death is one of them. Silencing of the necroptosis initiator Ripk3 occurs in a wide variety of cancer types including melanoma. Little is known about the role of the necroptosis executioner MLKL in tumor development. Studies often indicate opposing roles for MLKL as a tumor-suppressing or a tumor-promoting protein. This study investigates the role of MLKL during melanoma initiation and progression using a tamoxifen-inducible melanoma mouse model driven by melanocyte-specific overexpression of mutated Braf and simultaneous deletion of Pten ( Braf V600E Pten −/− ). In this model we observed a clear sex difference: melanoma initiation and progression were faster in females mice. Mlkl deficiency in male mice resulted in a modest but significant reduction of nevi growth rate compared to the littermate control. In these mice, infiltration and expansion of melanoma cells in the inguinal lymph node were also modestly decreased. This is likely to be a consequence of the delay in nevi development. No significant difference was observed in the Mlkl -deficient condition in female mice in which melanoma development was faster. Overall, our results indicate that in this genetic model MLKL has a minor role during melanoma initiation and progression.
... Importantly, the established knockout lines for CXCL9 and CXCL10 carry passenger mutations derived from the 129 embryonic stem cells used in their generation. [42][43][44] Thus, the generation of CXCL11 KI mice provides an appropriate control line for these widely used lines. Further, our observation that steady-state immune development and antiviral responses are largely unaffected by the physiological expression of CXCL11 is consistent with work that has directly compared CXCL9 and CXCL10 knockout animals and observed distinct roles. ...
The chemokine receptor CXCR3 is expressed on immune cells to co‐ordinate lymphocyte activation and migration. CXCR3 binds three chemokine ligands, CXCL9, CXCL10, and CXCL11. These ligands display distinct expression patterns and ligand signaling biases; however, how each ligand functions individually and collaboratively is incompletely understood. CXCL9 and CXCL10 are considered pro‐inflammatory chemokines during viral infection, while CXCL11 may induce a tolerizing state. The investigation of the individual role of CXCL11 in vivo has been hampered as C57BL/6 mice carry several mutations which result in a null allele. Here, we used CRISPR/Cas9 to correct these mutations on a C57BL/6 background. We validated that CXCL11KI mice expressed CXCL11 protein in dendritic cells, spleen and lung. CXCL11KI mice were largely phenotypically indistinguishable to C57BL/6 mice, both at steady‐state and during two models of viral infection. While CXCL11 expression did not modify acute anti‐viral responses, this study provides a new tool to understand the role of CXCL11 in other experimental settings.
... These cells were implanted to a C57BL/6 blastocyst, the recipient, and following germline transmission and at least 15 generations of backcrossing, a SARM1deficient mouse on the C57BL/6 background was achieved. However, it is now well established that mice generated in this way retain donor-derived genetic material, termed passenger genes or passenger mutations, in the region flanking the targeted gene [28]. The probability of a gene being of donor origin depends on the proximity to the targeted locus and the number of generations of backcrosses to C57BL/6 mice. ...
SARM1 is a TIR-domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages, however the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone marrow-derived macrophages (BMDM) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared to wild type (WT). However, we found passenger genes which are derived from the 129 donor strain of mice that flank the Sarm1 locus confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1-/- mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1’s function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem, and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation, and has provided important new models to further explore SARM1 function.
... In this regard, it has to be considered that concepts purely inferred from genetic studies can be misleading in light of the fact that passenger mutations can impact on the phenotype of interest. 101 With the availability of efficient genome engineering methodologies to manipulate human primary T cells, it will be also be feasible to address some of these concepts directly in the human system. ...
The concept of non-self recognition through germ-line encoded pattern recognition receptors (PRRs) has been well-established for professional innate immune cells. However, there is growing evidence that also T cells employ PRRs and associated effector functions in response to certain non-self or damage signals. Inflammasomes constitute a special subgroup of PRRs that is hardwired to a signaling cascade that culminates in the activation of caspase-1. Active caspase-1 processes pro-inflammatory cytokines of the IL-1 family and also triggers a lytic programmed cell death pathway known as pyroptosis. An increasing body of literature suggests that inflammasomes are also functional in T cells. On the one hand, conventional inflammasome signaling cascades have been described that operate similarly to pathways characterized in innate immune cells. On the other hand, unconventional functions have been suggested, in which certain inflammasome components play a role in unrelated processes, such as cell fate decisions and functions of T helper cells. In this review, we discuss our current knowledge on inflammasome functions in T cells and the biological implications of these findings for health and disease.
... Furthermore, genetically modified mice created using 129-derived embryonic stem cells often contain passenger mutations in sequences flanking targeted genes, hence contaminating the phenotypic outcome of genetic manipulation. For example, knockouts of several MMP genes located on mouse chromosome 9 (mmp3, mmp7, mmp8) contain passenger mutation that inactivates the gene encoding caspase 11, which is also a protease [28]. As a result, partial inactivation of caspase 11 may confound phenotypic interpretation of MMP genetic knockout models or perturb the identification of MMP substrates by mass spectrometry-assisted degradomic studies. ...
Long-term synaptic plasticity is shaped by the controlled reorganization of the synaptic proteome. A key component of this process is local proteolysis performed by the family of extracellular matrix metalloproteinases (MMPs). In recent years, considerable progress was achieved in identifying extracellular proteases involved in neuroplasticity phenomena and their protein substrates. Perisynaptic metalloproteinases regulate plastic changes at synapses through the processing of extracellular and membrane proteins. MMP9 was found to play a crucial role in excitatory synapses by controlling the NMDA-dependent LTP component. In addition, MMP3 regulates the L-type calcium channel-dependent form of LTP as well as the plasticity of neuronal excitability. Both MMP9 and MMP3 were implicated in memory and learning. Moreover, altered expression or mutations of different MMPs are associated with learning deficits and psychiatric disorders, including schizophrenia, addiction, or stress response. Contrary to excitatory drive, the investigation into the role of extracellular proteolysis in inhibitory synapses is only just beginning. Herein, we review the principal mechanisms of MMP involvement in the plasticity of excitatory transmission and the recently discovered role of proteolysis in inhibitory synapses. We discuss how different matrix metalloproteinases shape dynamics and turnover of synaptic adhesome and signal transduction pathways in neurons. Finally, we discuss future challenges in exploring synapse- and plasticity-specific functions of different metalloproteinases.
... For instance, the Ripk3 KD-KI +/+ and the Mlkl +/+ mice are more susceptible than Ripk3 +/+ for the same viral challenge dose (0.2x LD 50 ). The use of littermate controls is essential when comparing the outcome of an immunity-related stimulus between genetically modified strains 26,27 . Particularly when evaluating viral susceptibility of KO mice, specific littermate controls should be used in each experiment. ...
RIPK3 was reported to play an important role in the protection against influenza A virus (IAV) in vivo. Here we show that the requirement of RIPK3 for protection against IAV infection in vivo is only apparent within a limited dose range of IAV challenge. We found that this protective outcome is independent from RIPK3 kinase activity and from MLKL. This shows that platform function of RIPK3 rather than its kinase activity is required for protection, suggesting that a RIPK3 function independent of necroptosis is implicated. In line with this finding, we show that FADD-dependent apoptosis has a crucial additional effect in protection against IAV infection. Altogether, we show that RIPK3 contributes to protection against IAV in a narrow challenge dose range by a mechanism that is independent of its kinase activity and its capacity to induce necroptosis.
... Here, using RAG-2/MMP-13 double null mice, our data show that stromal MMP-13 contributes to myeloma-induced bone destruction by regulating the bioavailability of non-matrix molecules such as CXCL7 that impact osteoclast recruitment, formation and function as opposed to processing type I collagen. It is also important to note that several MMP-deficient mice carry a caspase-11 passenger mutation, including the MMP-13-null mice used in this study [50]. However, the in vitro and in vivo data obtained with our MMP-13-selective inhibitor support, in large part, those obtained with the RAG-2/ MMP-13-null mice indicating negligible if any caspase-11 contribution. ...
Multiple myeloma promotes systemic skeletal bone disease that greatly contributes to patient morbidity. Resorption of type I collagen–rich bone matrix by activated osteoclasts results in the release of sequestered growth factors that can drive progression of the disease. Matrix metalloproteinase-13 (MMP13) is a collagenase expressed predominantly in the skeleton by mesenchymal stromal cells (MSC) and MSC-derived osteoblasts. Histochemical analysis of human multiple myeloma specimens also demonstrated that MMP13 largely localizes to the stromal compartment compared with CD138+ myeloma cells. In this study, we further identified that multiple myeloma induces MMP13 expression in bone stromal cells. Because of its ability to degrade type I collagen, we examined whether bone stromal–derived MMP13 contributed to myeloma progression. Multiple myeloma cells were inoculated into wild-type or MMP13–null mice. In independent in vivo studies, MMP13–null mice demonstrated significantly higher overall survival rates and lower levels of bone destruction compared with wild-type controls. Unexpectedly, no differences in type I collagen processing between the groups were observed. Ex vivo stromal coculture assays showed reduced formation and activity in MMP13–null osteoclasts. Analysis of soluble factors from wild-type and MMP13–null MSCs revealed decreased bioavailability of various osteoclastogenic factors including CXCL7. CXCL7 was identified as a novel MMP13 substrate and regulator of osteoclastogenesis. Underscoring the importance of host MMP13 catalytic activity in multiple myeloma progression, we demonstrate the in vivo efficacy of a novel and highly selective MMP13 inhibitor that provides a translational opportunity for the treatment of this incurable disease.
Significance
Genetic and pharmacologic approaches show that bone stromal–derived MMP13 catalytic activity is critical for osteoclastogenesis, bone destruction, and disease progression.
... Our findings differ from some previous reports [42,63], although the use of littermates was not reported in these studies. This is an important consideration, because even small genetic differences, such as those between C57BL/6 J and C57BL/6NJ mouse strains, were reported to impact susceptibility of the latter to TNFor LPS-induced sterile sepsis [64]. Each of the models that we employed are epithelial tumor models, driven by specific aberrations in the WNT-signaling pathway [49], which mimic the first step of CRC development in humans. ...
Chronic inflammation of the large intestine is associated with an increased risk of developing colorectal cancer (CRC), the second most common cause of cancer-related deaths worldwide. Necroptosis has emerged as a form of lytic programmed cell death that, distinct from apoptosis, triggers an inflammatory response. Dysregulation of necroptosis has been linked to multiple chronic inflammatory diseases, including inflammatory bowel disease and cancer. Here, we used murine models of acute colitis, colitis-associated CRC, sporadic CRC, and spontaneous intestinal tumorigenesis to investigate the role of necroptosis in these gastrointestinal pathologies. In the Dextran Sodium Sulfate-induced acute colitis model, in some experiments, mice lacking the terminal necroptosis effector protein, MLKL, or its activator RIPK3, exhibited greater weight loss compared to wild-type mice, consistent with some earlier reports. However, the magnitude of weight loss and accompanying inflammatory pathology upon Mlkl deletion varied substantially between independent repeats. Such variation provides a possible explanation for conflicting literature reports. Furthermore, contrary to earlier reports, we observed that genetic deletion of MLKL had no impact on colon cancer development using several mouse models. Collectively, these data do not support an obligate role for necroptosis in inflammation or cancer within the gastrointestinal tract.
... In the current study we show, using in silico modelling, how these substitutions affect the interaction with BACE1. We have also used CRISPR-Cas9 technology to scarlessly humanize the endogenous Aβ sequence in the mouse and rat App genes [15] and investigate the effects on APP processing. We also created a PSEN1 knock-in mutation to generate a rat model for AD. ...
Background
Three amino acid differences between rodent and human APP affect medically important features, including β-secretase cleavage of APP and Aβ peptide aggregation (De Strooper et al., EMBO J 14:4932-38, 1995; Ueno et al., Biochemistry 53:7523-30, 2014; Bush, 2003, Trends Neurosci 26:207–14). Most rodent models for Alzheimer’s disease (AD) are, therefore, based on the human APP sequence, expressed from artificial mini-genes randomly inserted in the rodent genome. While these models mimic rather well various biochemical aspects of the disease, such as Aβ-aggregation, they are also prone to overexpression artifacts and to complex phenotypical alterations, due to genes affected in or close to the insertion site(s) of the mini-genes (Sasaguri et al., EMBO J 36:2473-87, 2017; Goodwin et al., Genome Res 29:494-505, 2019). Knock-in strategies which introduce clinical mutants in a humanized endogenous rodent APP sequence (Saito et al., Nat Neurosci 17:661-3, 2014) represent useful improvements, but need to be compared with appropriate humanized wildtype (WT) mice.
Methods
Computational modelling of the human β-CTF bound to BACE1 was used to study the differential processing of rodent and human APP. We humanized the three pivotal residues we identified G676R, F681Y and R684H (labeled according to the human APP770 isoform) in the mouse and rat genomes using a CRISPR-Cas9 approach. These new models, termed mouse and rat App hu/hu , express APP from the endogenous promotor. We also introduced the early-onset familial Alzheimer’s disease (FAD) mutation M139T into the endogenous Rat Psen 1 gene.
Results
We show that introducing these three amino acid substitutions into the rodent sequence lowers the affinity of the APP substrate for BACE1 cleavage. The effect on β-secretase processing was confirmed as both humanized rodent models produce three times more (human) Aβ compared to the original WT strain. These models represent suitable controls, or starting points, for studying the effect of transgenes or knock-in mutations on APP processing (Saito et al., Nat Neurosci 17:661-3, 2014). We introduced the early-onset familial Alzheimer’s disease (FAD) mutation M139T into the endogenous Rat Psen 1 gene and provide an initial characterization of Aβ processing in this novel rat AD model.
Conclusion
The different humanized APP models (rat and mouse) expressing human Aβ and PSEN1 M139T are valuable controls to study APP processing in vivo allowing the use of a human Aβ ELISA which is more sensitive than the equivalent system for rodents. These animals will be made available to the research community.
... CD163-deficient (CD163 −/− , C57BL/6N background) mice were purchased from the Knockout Mouse Project (https:// www.komp.org). Mice were backcrossed with C57BL/6N mice (CREA Japan, Shizuoka, Japan) to solve the problem of passenger mutations and SNPs observed in congenic mice, 41,42 and then CD163 +/− mice were housed under specific pathogen-free conditions. Mice were genotyped as previously described. ...
Objectives
Septic (or endotoxin) shock is a severe systemic inflammatory disease caused by bacteraemia or endotoxaemia. Although it is known that increased serum levels of CD163 are observed in septic/endotoxin shock patients, the exact function and significance of CD163 in macrophage activation remain unclear. Therefore, in the current study, we tested whether CD163 contributes to the pathogenesis of endotoxin shock in mice.
Methods and results
In samples obtained from autopsy, the number of CD163‐positive macrophages was increased in the kidney, liver, heart, bone marrow and spleen of patients who had died from septic/endotoxin shock when compared to patients who had died from other causes. The animal study revealed a significantly lower survival rate in CD163‐deficient mice after lipopolysaccharide (LPS) injection. Several cytokines and oxidative stress‐related molecules were significantly elevated in the sera of LPS‐induced endotoxin shock mice models. Higher concentrations of IL‐6, TNF‐α, IL‐1β, nitrite () and nitrate () and a lower concentration of IL‐10 were observed in CD163‐deficient mice treated with LPS. Similar results were observed in CD163‐deficient LPS‐stimulated macrophages. Furthermore, in an antitype II collagen antibody‐induced arthritis (CAIA), rheumatoid arthritis model, inflammation and bone erosion scores as well as the expression of IL‐6 and IL‐1β were significantly increased in CD163‐deficient mice.
Conclusions
CD163 was suggested to be involved in the regulation of inflammatory cytokine expression in septic/endotoxin shock and CAIA.
... Lastly, an important caveat for congenic GEMM mouse strains that should be mentioned is that they appear to harbor significant amounts of passenger mutations as a consequence of genetic variation from embryonic stem cells (ESCs) used to establish the mouse lines [119]. While this likely poses less of an issue for the vast majority of translational cancer mouse studies, this could have the potential to interfere with interpretation of results in studies using congenic mice particularly as it relates to studies focused on tumor heterogeneity and identification of driver genes. ...
Recent developments in pre-clinical screening tools, that more reliably predict the clinical effects and adverse events of candidate therapeutic agents, has ushered in a new era of drug development and screening. However, given the rapid pace with which these models have emerged, the individual merits of these translational research tools warrant careful evaluation in order to furnish clinical researchers with appropriate information to conduct pre-clinical screening in an accelerated and rational manner. This review assesses the predictive utility of both well-established and emerging pre-clinical methods in terms of their suitability as a screening platform for treatment response, ability to represent pharmacodynamic and pharmacokinetic drug properties, and lastly debates the translational limitations and benefits of these models. To this end, we will describe the current literature on cell culture, organoids, in vivo mouse models, and in silico computational approaches. Particular focus will be devoted to discussing gaps and unmet needs in the literature as well as current advancements and innovations achieved in the field, such as co-clinical trials and future avenues for refinement.
... Besides, during the generation of genetically modified congenic mice, the targeted gene is flanked by passenger genes that are originated from the donor strain. These passenger genes, usually harboring mutations *Some of the phenotypes may appear only in some substrains (passenger mutations), will still be retained in the congenic mice even after intensive backcrossing [262]. All of these could contribute to unexpected phenotypes and reduce the reproducibility of a particular mouse strain. ...
The laboratory mouse is the most commonly used mammalian model for biomedical research. An enormous number of mouse models, such as gene knockout, knockin, and overexpression transgenic mice, have been created over the years. A common practice to maintain a genetically modified mouse line is backcrossing with standard inbred mice over several generations. However, the choice of inbred mouse for backcrossing is critical to phenotypic characterization because phenotypic variabilities are often observed between mice with different genetic backgrounds. In this review, the major features of commonly used inbred mouse lines are discussed. The aim is to provide information for appropriate selection of inbred mouse lines for genetic and behavioral studies.
Background
The channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood.
Methods
Wild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice.
Results
Global or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca²⁺ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG.
Conclusions
The present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40779-024-00525-8.
Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a mouse model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC infected SLPI-deficient (Slpi -/- ) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi +/+ ) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi -/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with history of recent or recurrent UTI (rUTI), suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI protects against acute UTI in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.
Pannexin 1 (PANX1) is a widely expressed large-pore ion channel located in the plasma membrane of almost all vertebrate cells. It possesses a unique ability to act as a conduit for both inorganic ions (e.g. potassium or chloride) and bioactive metabolites (e.g. ATP or glutamate), thereby activating varying signaling pathways in an autocrine or paracrine manner. Given its crucial role in cell-cell interactions, the activity of PANX1 has been implicated in maintaining homeostasis of cardiovascular, immune, and nervous systems. Dysregulation of PANX1 has also been linked to numerous diseases, such as ischemic stroke, seizure, and inflammatory disorders. Therefore, the mechanisms underlying different modes of PANX1 activation and its context-specific channel properties have gathered significant attention. In this review, we summarize the roles of PANX1 in various physiological processes and diseases, and analyze the accumulated lines of evidence supporting diverse molecular mechanisms associated with different PANX1 activation modalities. We focus on examining recent discoveries regarding PANX1 regulations by reversible post-translational modifications, elevated intracellular calcium concentration, and protein-protein interactions, as well as by irreversible cleavage of its C-terminal tail. Additionally, we delve into the caveats in the proposed PANX1 gating mechanisms and channel open-closed configurations by critically analyzing the structural insights derived from cryo-EM studies and the unitary properties of PANX1 channels. By doing so, we aim to identify potential research directions for a better understanding of the functions and regulations of PANX1 channels.
Inflammasomes are cytoplasmic organelles that stimulate inflammation upon cellular detection of infectious or non-infectious stress. While much foundational work has focused on the infection-associated aspects of inflammasome activities, recent studies have highlighted the role of inflammasomes in non-infectious cellular and organismal functions. Herein, we discuss the evolution of inflammasome components and highlight characteristics that permit inflammasome regulation of physiologic processes. We focus on emerging data that highlight the importance of inflammasome proteins in the regulation of reproduction, development, and malignancy. A framework is proposed to contextualize these findings.
Regulated cell death is an inherent attribute common to all metazoans. In animals, such as humans, two fundamentally different types of cell death are recognized: apoptosis and necrosis. The main distinction is that apoptosis is a silent demise without inflammatory consequences, whereas necrosis is highly inflammatory. Necrotic cell death encompasses the pathways known as necroptosis and pyroptosis. In this article we describe the biochemical players, regulation, and execution of these highly studied mechanisms of cell death in health and disease.
The development and regeneration of skeletal muscle are mediated by satellite cells (SCs), which ensure the efficient formation of myofibers while repopulating the niche that allows muscle repair following injuries. Pannexin 1 (Panx1) channels are expressed in SCs and their levels increase during differentiation in vitro, as well as during skeletal muscle development and regeneration in vivo. Panx1 has recently been shown to regulate muscle regeneration by promoting bleb‐based myoblast migration and fusion. While skeletal muscle is largely influenced in a sex‐specific way, the sex‐dependent roles of Panx1 in regulating skeletal muscle and SC function remain to be investigated. Here, using global Panx1 knockout (KO) mice, we demonstrate that Panx1 loss reduces muscle fiber size and strength, decreases SC number, and alters early SC differentiation and myoblast fusion in male, but not in female mice. Interestingly, while both male and female Panx1 KO mice display an increase in the number of regenerating fibers following acute injury, the newly formed fibers in male Panx1 KO mice are smaller. Overall, our results demonstrate that Panx1 plays a significant role in regulating muscle development, regeneration, and SC number and function in male mice and reveal distinct sex‐dependent functions of Panx1 in skeletal muscle.
Regulated cell death is defined as genetically encoded pathways that lead towards the demise of cells. In mammals, cell demise can be either inflammatory or non-inflammatory, depending on whether the mechanism of death results in cell rupture or not. Inflammatory cell death can lead towards acute and chronic disease. Therefore, it becomes important to distinguish the mechanisms that result in these different inflammatory cell death outcomes. Apoptosis is a non-inflammatory form of cell death where cells resist rupture. In contrast, pyroptosis and necroptosis are inflammatory forms of cell death principally because of release of pro-inflammatory mediators from cells undergoing lysis. This review focusses on the mechanisms of these different cell death outcomes with specific emphasis on the caspase family of proteolytic enzymes.
Homotypic signaling lymphocyte activation molecule (SLAM) receptor–ligand cell surface interactions between myeloid and lymphoid cells regulate innate and adaptive immune responses. In this article, we report that SLAMF1 is indispensable for host resistance to primary and vaccine-induced protection against fungal infection. Because vaccine immunity is dependent on cell-mediated immunity, we investigated the development of Ag-specific T cells. We studied the T cell–intrinsic and –extrinsic role of SLAMF1. We generated SLAMF1−/− TCR transgenic mice and analyzed the responses of adoptively transferred T cells. We also tracked endogenous Ag-specific T cells by using a tetramer. Intrinsic and extrinsic SLAMF1 signaling was dispensable for the development of antifungal Th1 and Th17 cells, which are requisite for the acquisition of vaccine-induced immunity. Despite intact T cell development, vaccinated SLAMF1−/− mice failed to control fungal infection. Failed accumulation of Ag-specific T cells in the lung on infection of vaccinated mice was due to uncontrolled early infection and inflammation, revealing a role for SLAMF1 in innate host immunity.
Exposure to high dose radiation causes life-threatening acute and delayed effects. Defining the mechanisms of lethal radiation-induced acute toxicity of gastrointestinal and hematopoietic tissues are critical steps to identify drug targets to mitigate and protect against the acute radiation syndrome (ARS). For example, one rational approach would be to design pharmaceuticals that block cell death pathways to preserve tissue integrity in radiation-sensitive organ systems including the gastrointestinal tract and hematopoietic compartment. A previous study reported that the inflammasome pathway, which mediates inflammatory cell death through pyroptosis, promotes ARS. However, we show that mice lacking the inflammatory executioner caspases, caspase-1 and caspase-11, are not protected from ARS when compared directly to littermates expressing caspase-1 and caspase-11. These results suggest that alternative pathways will need to be targeted by drugs that successfully mitigate and protect against the ARS.
This chapter is an overview of the current and growing knowledge of the genetics of laboratory rodents, specifically the mouse (Mus musculus) and the rat (Rattus norvegicus), the two main species used in biomedical research. We present basic information about Mendelian genetics and on the structure of the mouse and rat genomes, including the protein-coding DNA and the more intriguing non-coding DNA sequences, abundant in repetitive DNA, transposable elements and different types of genetic polymorphisms. Experiments should be performed with carefully designed and approved protocols, including the use of genetically defined animals. Thus, in this chapter we discussed the different types of genetically standardized laboratory strains and the aspects related to their genetic quality control. We also present the different types of genetically altered mice and rats, including spontaneous and chemically induced mutations, random transgenesis, targeted mutagenesis using embryonic stem cells and the novel genome editing techniques. It is very important for the veterinarians and technicians in charge of animal facilities, as well as for researchers and students using mouse and rat models that they have an available up-to-date information devoted to the genetics of these species.
Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Despite their major impact on public health and economy, little is known about how HuNoVs interact with their host during infection. Murine norovirus (MNV) is genetically related to HuNoVs and is commonly used to study norovirus-host interactions and basic aspects of norovirus biology. In this dissertation, we aimed to characterize the initial steps of MNV infection at the host and cellular levels. Microfold (M) cells are specialized intestinal epithelial cells for transporting lumenal antigens across the epithelial barrier by transcytosis. We are interested in the role M cells play in MNV infection in vivo. Previous data from our lab showed that oral MNV infection is significantly reduced in BALB/c mice depleted of M cells and absent in Rag2null/γcnull mice. To expand the study on the role of M cells in MNV infection, we used a conditional knockout mouse model of M cell deficiency (C57BL/6 background). Unlike our previous findings, mice deficient in M cells (M-less mice) and littermates had similar MNV-1 and MNV.CR3 yields, suggesting M cells are not required during MNV infection in this model. However, M-less mice infected with MNV-1 had significantly reduced viral titers in the MLN, which suggests that trafficking of MNV-1 from the intestinal lumen to the MLN is M cell-dependent. A key determinant of noroviruses’ cell tropism and species-specificity is expression of host cellular attachment/entry receptors. We investigated whether host cellular proteins play a role during the early steps of norovirus infection. Using VOPBA followed by MS/MS analysis in two permissive cell lines, RAW264.7 (macrophage-like) and SRDC (DC-like), four cellular membrane proteins were identified as candidates. Loss-of-function studies revealed that CD36 and CD44 promoted MNV-1 binding to primary dendritic cells, while CD98 and transferrin receptor 1 (TfRc) facilitated MNV-1 binding to RAW 264.7 cells. Furthermore, MNV-1 P domain interacted directly with the extracellular domains of recombinant CD36, CD98 and TfRc by ELISA. Additionally, CD98 may play a role in post-binding stages of MNV-1 infection. Our studies demonstrated that multiple membrane proteins can promote efficient MNV-1 infection in a cell type-specific manner.
Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.
Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.
The integration of neurons into networks relies on the formation of dendritic spines. These specialized structures arise from dynamic filopodia-like dendritic protrusions. It was recently reported that cortical neurons lacking the channel protein pannexin 1 (PANX1) exhibited higher dendritic spine densities. Here, we expanded on those findings to investigate, at an earlier developmental time point (with more abundant dendritic protrusions), whether differences in the properties of dendritic protrusion dynamics could contribute to this previously discovered phenomenon. Using a fluorescent membrane tag (mCherry-CD9-10) to visualize dendritic protrusions in developing neurons [at 10 d in vitro (DIV10)], we confirmed that lack of PANX1 led to higher protrusion density, while transient transfection of Panx1 led to decreased protrusion density. To quantify the impact of PANX1 expression on protrusion formation, elimination, and motility, we used live cell imaging in DIV10 neurons (one frame every 5 s for 10 min). We discovered that at DIV10, loss of PANX1 stabilized protrusions. Notably, re-expression of PANX1 in Panx1 knock-out (KO) neurons resulted in a significant increase in protrusion motility and turnover. In summary, these new data revealed that PANX1 could regulate the development of dendritic spines, in part, by controlling dendritic protrusion dynamics.
Increasing evidence suggests that functional impairments at the level of the neurovascular unit (NVU) underlie many neurodegenerative and neuroinflammatory diseases. While being part of the NVU, astrocytes have been largely overlooked in this context and only recently, tightening of the glia limitans has been put forward as an important neuroprotective response to limit these injurious processes. In this study, using the retina as a central nervous system (CNS) model organ, we investigated the structure and function of the glia limitans, and reveal that the blood–retina barrier and glia limitans function as a coordinated double barrier to limit infiltration of leukocytes and immune molecules. We provide in vitro and in vivo evidence for a protective response at the NVU upon CNS injury, which evokes inflammation‐induced glia limitans tightening. Matrix metalloproteinase‐3 (MMP‐3) was found to be a crucial regulator of this process, thereby revealing its beneficial and immunomodulatory role in the CNS. in vivo experiments in which MMP‐3 activity was deleted via genetic and pharmacological approaches, combined with a comprehensive study of tight junction molecules, glial end feet markers, myeloid cell infiltration, cytokine expression and neurodegeneration, show that MMP‐3 attenuates neuroinflammation and neurodegeneration by tightening the glia limitans, thereby pointing to a prominent role of MMP‐3 in preserving the integrity of the NVU upon injury. Finally, we gathered promising evidence to suggest that IL1b, which is also regulated by MMP‐3, is at least one of the molecular messengers that induces glia limitans tightening in the injured CNS. In the neurovascular unit, the blood–retinal barrier and glia limitans function as a coordinated double barrier to limit infiltration of leukocytes. MMP‐3 contributes to preserving the integrity of the retinal neurovascular unit upon optic nerve crush by attenuating neuroinflammation and neurodegeneration.
Background
The inbred mouse strain C57BL/6 is widely used in both models of addiction and immunological disease. However, there are pronounced phenotypic differences in ethanol consumption and innate immune response between B6 substrains. The focus of this study was to examine the effects of substrain on innate immune response and neuroimmune‐induced escalation of voluntary ethanol consumption. The main goal was to identify if substrain differences in immune response can account for differences in ethanol behavior.
Methods
We compared acute innate immune response to a viral dsRNA mimic, polyinosinic:polycytidylic acid (poly(I:C)), in brain using qRT‐PCR in both C57BL/6N and C57BL/6J mice. Next, we used a neuroimmune model of escalation using poly(I:C) to compare drinking behavior between substrains. Finally, we compared brain neuroimmune response to both ethanol and repeated poly(I:C) in both strains as a way to account for differences in ethanol behavior.
Results
We found that C57BL/6 strains have differing immune response and drinking behaviors. C57BL/6N mice have a shorter but more robust inflammatory response to acute poly(I:C). In contrast, C57BL/6J mice have a smaller but longer lasting acute immune response to poly(I:C). In our neuroimmune‐induced escalation model, C57BL/6J mice but not C57BL/6N mice escalate ethanol intake after poly(I:C). Finally, only C57BL/6J mice show enhanced proinflammatory transcript abundance after poly(I:C) and ethanol, suggesting that longer lasting immune responses are critical to neuroimmune drinking phenotypes.
Conclusions
Altogether, this work has elucidated additional influences that substrain has on both innate immune response and drinking phenotypes. Our observations highlight the importance of considering and reporting the source and background used for production of transgenic and knockout mice. This data provides further evidence that genetic background must be carefully considered when investigating the role of neuroimmune signaling in ethanol abuse.
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that form a family of 24 members in mammals. Evidence of the pathological roles of MMPs in various diseases, combined with their druggability, has made them attractive therapeutic targets. Initial drug discovery efforts focused on the roles of MMPs in cancer progression, and more than 50 MMP inhibitors have been investigated in clinical trials in various cancers. However, all of these trials failed. Reasons for failure include the lack of inhibitor specificity and insufficient knowledge about the complexity of the disease biology. MMPs are also known to be involved in several inflammatory processes, and there are new therapeutic opportunities for MMP inhibitors to treat such diseases. In this Review, we discuss the recent advances made in understanding the role of MMPs in inflammatory diseases and the therapeutic potential of MMP inhibition in those conditions.
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
Disrupted in schizophrenia-1 (DISC1) gene is associated with several neuropsychiatric disorders as it is disrupted by a balanced translocation involving chromosomes 1 and 11 in a large Scottish pedigree with high prevalence of schizophrenia, bipolar disorder and major depression. Since its identification, several mouse models with DISC1 genetic modifications have been generated using different approaches. Interestingly, a natural deletion of 25bp in the 129 mouse strain alters the DISC1 gene reading frame leading to a premature stop codon very close to the gene breakpoint in the mutant allele of the Scottish family. In the present study we confirmed that the 129DISC1(Del) mutation results in reduced level of full length DISC1 in hippocampus of heterozygous mice and we have characterized the behavioral consequences of heterozygous 129DISC1(Del) mutation in a mixed B6129 genetic background. We found alterations in spontaneous locomotor activity (hyperactivity in males and hypoactivity in females), deficits in pre-pulse inhibition (PPI) and also increased despair behavior in heterozygous 129DISC1(Del) mice, thus reproducing typical behaviors associated to psychiatric disorders. Since this mouse strain is widely and commercially available, we propose it as an amenable tool to study DISC1-related biochemical alterations and psychiatric behaviors.
Significance
Ras oncogens have been implicated in high proportion of cancers. However, selective treatment for Ras oncogenes is not yet available and components that could mediate Ras functions are being extensively assessed as alternative targets. For some potential targets, such as phospholipase Cε (PLCε), it is not clear how they contribute to tumor development. Here we provide several lines of experimental evidence that support a tumor suppressor function of PLCε, contrasting a positive role of several other well-defined effectors of Ras. Our results show a tumor suppressor role for this PLC in animal models and describe its impact on cell proliferation. Together with observations of marked down-regulation in several types of tumors, this study suggests its use as a biological marker in cancer.
Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiological alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly-publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without over-interpretation. Hopefully a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.
The serpin family comprises of a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease.
Matrix metalloproteinase 7 (MMP7) is a member of the MMP family. In the small intestine, MMP7 is responsible for activating α-defensins, which are broad-spectrum anti-microbial peptides produced by the Paneth cells. We report that MMP7(-/-) mice are resistant to LPS-induced lethality and that this resistance is correlated with reduced levels of systemic cytokines. LPS induced the upregulation and activation of MMP7 in the small intestine, degranulation of the Paneth cells, and induction of intestinal permeability in MMP7(+/+) mice. In MMP7(-/-) mice, both LPS-induced intestinal permeability and consequent bacterial translocation to the mesenteric lymph nodes were reduced. Based on gene expression analysis and evaluation of intestinal damage, we attribute the protected state of MMP7(-/-) mice to reduced intestinal inflammation. Interestingly, we found that different α-defensins, namely Crp1 (DEFA1) and Crp4 (DEFA4), can stimulate IL-6 release in macrophages and ileum explants in a TLR4 independent way. We conclude that absence of MMP7 protects mice from LPS-induced intestinal permeability and lethality, and suggest that MMP7-activated α-defensins, in addition to their previously recognized bactericidal and anti-inflammatory roles, may exhibit pro-inflammatory activities in the intestine by activating macrophages and amplifying the local inflammatory response in the gut, leading to intestinal leakage and subsequent increase in systemic inflammation.Mucosal Immunology advance online publication, 16 October 2013; doi:10.1038/mi.2013.76.
Move Over, TLR4
The innate immune system senses bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) (see the Perspective by Kagan ). However, Kayagaki et al. (p 1246 , published online 25 July) and Hagar et al. (p. 1250 ) report that the hexa-acyl lipid A component of LPS from Gramnegative bacteria is able to access the cytoplasm and activate caspase-11 to signal immune responses independently of TLR4. Mice that lack caspase-11 are resistant to LPS-induced lethality, even in the presence of TLR4.
The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as The International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.
We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.
Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.
Several pathological processes, such as sepsis and inflammatory bowel disease (IBD), are associated with impairment of intestinal epithelial barrier. Here, we investigated the role of matrix metalloproteinase MMP13 in these diseases. We observed that MMP13(-/-) mice display a strong protection in LPS- and caecal ligation and puncture-induced sepsis. We could attribute this protection to reduced LPS-induced goblet cell depletion, endoplasmic reticulum stress, permeability and tight junction destabilization in the gut of MMP13(-/-) mice compared to MMP13(+/+) mice. Both in vitro and in vivo, we found that MMP13 is able to cleave pro-TNF into bioactive TNF. By LC-MS/MS, we identified three MMP13 cleavage sites, which proves that MMP13 is an alternative TNF sheddase next to the TNF converting enzyme TACE. Similarly, we found that the same mechanism was responsible for the observed protection of the MMP13(-/-) mice in a mouse model of DSS-induced colitis. We identified MMP13 as an important mediator in sepsis and IBD via the shedding of TNF. Hence, we propose MMP13 as a novel drug target for diseases in which damage to the gut is essential.
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate
RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases
can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also
be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple
guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian
genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
With the effort of the International Phenotyping Consortium to produce thousands of strains with conditional potential gathering steam, there is growing recognition that it must be supported by a rich toolbox of cre driver strains. The approaches to build cre strains have evolved in both sophistication and reliability, replacing first-generation strains with tools that can target individual cell populations with incredible precision and specificity. The modest set of cre drivers generated by individual labs over the past 15+ years is now growing rapidly, thanks to a number of large-scale projects to produce new cre strains for the community. The power of this growing resource, however, depends upon the proper deep characterization of strain function, as even the best designed strain can display a variety of undesirable features that must be considered in experimental design. This must be coupled with the parallel development of informatics tools to provide functional data to the user and facilitated access to the strains through public repositories. We discuss the current progress on all of these fronts and the challenges that remain to ensure the scientific community can capitalize on the tremendous number of mouse resources at their disposal.
Systemic inflammatory response syndrome (SIRS) is a highly mortal inflammatory disease, associated with systemic inflammation and organ dysfunction. SIRS can have a sterile cause or can be initiated by an infection, called sepsis. The prevalence is high, and available treatments are ineffective and mainly supportive. Consequently, there is an urgent need for new treatments. The brain is one of the first organs affected during SIRS, and sepsis and the consequent neurological complications, such as encephalopathy, are correlated with decreased survival. The choroid plexus (CP) that forms the blood-CSF barrier (BCSFB) is thought to act as a brain "immune sensor" involved in the communication between the peripheral immune system and the CNS. Nevertheless, the involvement of BCSFB integrity in systemic inflammatory diseases is seldom investigated. We report that matrix metalloprotease-8 (MMP8) depletion or inhibition protects mice from death and hypothermia in sepsis and renal ischemia/reperfusion. This effect could be attributed to MMP8-dependent leakage of the BCSFB, caused by collagen cleavage in the extracellular matrix of CP cells, which leads to a dramatic change in cellular morphology. Disruption of the BCSFB results in increased CSF cytokine levels, brain inflammation, and downregulation of the brain glucocorticoid receptor. This receptor is necessary for dampening the inflammatory response. Consequently, MMP8(+/+) mice, in contrast to MMP8(-/-) mice, show no anti-inflammatory response and this results in high mortality. In conclusion, we identify MMP8 as an essential mediator in SIRS and, hence, a potential drug target. We also propose that the mechanism of action of MMP8 involves disruption of the BCSFB integrity.
Ditching Invading DNA
Bacteria and archaea protect themselves from invasive foreign nucleic acids through an RNA-mediated adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas). Jinek et al. (p. 816 , published online 28 June; see the Perspective by Brouns ) found that for the type II CRISPR/Cas system, the CRISPR RNA (crRNA) as well as the trans-activating crRNA—which is known to be involved in the pre-crRNA processing—were both required to direct the Cas9 endonuclease to cleave the invading target DNA. Furthermore, engineered RNA molecules were able to program the Cas9 endonuclease to cleave specific DNA sequences to generate double-stranded DNA breaks.
Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.
A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117-121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1(-/-) animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context.
Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11(-/-) mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1(-/-) mice lack both caspase-11 and caspase-1. Interestingly, Casp11(-/-) macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1(-/-)Casp11(129mt/129mt) macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.
Structural variation is widespread in mammalian genomes and is an important cause of disease, but just how abundant and important structural variants (SVs) are in shaping phenotypic variation remains unclear. Without knowing how many SVs there are, and how they arise, it is difficult to discover what they do. Combining experimental with automated analyses, we identified 711,920 SVs at 281,243 sites in the genomes of thirteen classical and four wild-derived inbred mouse strains. The majority of SVs are less than 1 kilobase in size and 98% are deletions or insertions. The breakpoints of 160,000 SVs were mapped to base pair resolution, allowing us to infer that insertion of retrotransposons causes more than half of SVs. Yet, despite their prevalence, SVs are less likely than other sequence variants to cause gene expression or quantitative phenotypic variation. We identified 24 SVs that disrupt coding exons, acting as rare variants of large effect on gene function. One-third of the genes so affected have immunological functions.
We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
Metabolic syndrome is a group of obesity-related metabolic abnormalities that increase an individual’s risk of developing
type 2 diabetes and cardiovascular disease. Here, we show that
mice genetically deficient in Toll-like receptor 5 (TLR5), a component of the innate immune system that is expressed in the
gut mucosa and that helps defend against infection, exhibit hyperphagia and develop hallmark features of metabolic syndrome,
including hyperlipidemia, hypertension, insulin resistance, and increased adiposity. These metabolic changes correlated with
changes in the composition of the gut microbiota, and transfer of the gut microbiota from TLR5-deficient mice to wild-type
germ-free mice conferred many features of metabolic syndrome to the recipients. Food restriction prevented obesity, but not
insulin resistance, in the TLR5-deficient mice. These results support the emerging view that the gut microbiota contributes
to metabolic disease and suggest that malfunction of the innate immune system may promote the development of metabolic syndrome.
The mammalian immune system is optimized to cope effectively with the constant threat of pathogens. However, when the immune system overreacts, sepsis, severe sepsis, or septic shock can develop. Despite extensive research, these conditions remain the leading cause of death in intensive care units. The matrix metalloproteinases (MMPs) constitute a family of proteases that are expressed in developmental, physiological, and pathological processes and also in response to infections. Studies using MMP inhibitors and MMP knockout mice indicate that MMPs play essential roles in infection and in the host defense against infection. This review provides a brief introduction to some basic concepts of infections caused by gram-negative bacteria and reviews reports describing MMP expression and inhibition, as well as studies with MMP-deficient mice in models of infection caused by gram-negative bacteria and of septic shock. We discuss whether MMPs should be considered novel drug targets in infection and septic shock.
The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source
of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels
of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal
in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes
retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2−/− mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout
model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian
gland and skin. The Harderian gland's transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent
genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually
immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after
UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation
of genes involved in hypertrophic responses.
To evaluate the effect of genetic background on high-density lipoprotein cholesterol (HDL) levels in Soat1(-/-) mice, we backcrossed sterol O-acyltransferase 1 (Soat1)(-/-) mice, originally reported to have elevated HDL levels, to C57BL/6 mice and constructed a congenic strain with only a small region (3.3Mb) of 129 alleles, specifically excluding the nearby apolipoprotein A-II (Apoa2) gene from 129. HDL levels in these Soat1(-/-) mice were no different from C57BL/6, indicating that the passenger gene Apoa2 caused the previously reported elevation of HDL in these Soat1(-/-) mice. Because many knockouts are made in strain 129 and then subsequently backcrossed into C57BL/6, it is important to identify quantitative trait loci (QTL) that differ between 129 and C57BL/6 so that one can guard against effects ascribed to a knockout but really caused by a passenger gene from 129. To provide such data, we generated 528 F(2) progeny from an intercross of 129S1/SvImJ and C57BL/6 and measured HDL concentrations in F(2) animals first fed chow and then atherogenic diet. A genome wide scan using 508 single-nucleotide polymorphisms (SNPs) identified 19 QTL, 2 of which were male specific and 2 were female specific. Using comparative genomics and haplotype analysis, we narrowed QTL on chromosomes 3, 5, 8, 17, and 18 to 0.5, 6.3, 2.6, 1.1, and 0.6 Mb, respectively. These data will serve as a reference for any effort to test the impact of candidate genes on HDL using a knockout strategy.
Gene targeting to create null mutations in mice is a powerful new tool in biology which will allow the molecular dissection of complex phenotypes such as mammalian brain function, and learning and memory. However, the attempt to interpret the phenotypical changes which arise in null-mutant mice is subject to several caveats. For example, the ability to disrupt a single gene in a targeted manner might lead one to overlook the effects of other genes. Ignoring the genetic background might lead to misinterpretation of results: the present article will demonstrate that the phenotypical abnormalities attributed to the null mutation in several molecular neurobiological studies could simply result from the effects of background genes.
Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a manner which could severely compromise the interpretation of the mutant phenotype. The present overview summarizes the literature on a wide variety of behavioral traits for the 129, C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field activity, learning and memory tasks, aggression, sexual and parental behaviors, acoustic startle and prepulse inhibition, and the behavioral actions of ethanol, nicotine, cocaine, opiates, antipsychotics, and anxiolytics. Using the referenced information, molecular geneticists can choose optimal parental strains of mice, and perhaps develop new embryonic stem cell progenitors, for new knockouts and transgenics to investigate gene function, and to serve as animal models in the development of novel therapeutics for human genetic diseases.
Excess tissue glucocorticoid action may underlie the dyslipidemia, insulin resistance, and impaired glucose tolerance of the metabolic syndrome. 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) catalyzes conversion of circulating inert 11-dehydrocorticosterone into active corticosterone, thus amplifying local intracellular glucocorticoid action, particularly in liver. The importance of 11beta-HSD-1 in glucose homeostasis is suggested by the resistance of 11beta-HSD-1(-/-) mice to hyperglycemia upon stress or obesity, due to attenuated gluconeogenic responses. The present study further investigates the metabolic consequences of 11beta-HSD-1 deficiency, focusing on the lipid and lipoprotein profile. Ad lib fed 11beta-HSD-1(-/-) mice have markedly lower plasma triglyceride levels. This appears to be driven by increased hepatic expression of enzymes of fat catabolism (carnitine palmitoyltransferase-I, acyl-CoA oxidase, and uncoupling protein-2) and their coordinating transcription factor, peroxisome proliferator-activated receptor-alpha (PPARalpha). 11beta-HSD-1(-/-) mice also have increased HDL cholesterol, with elevated liver mRNA and serum levels of apolipoprotein AI. Conversely, liver Aalpha-fibrinogen mRNA levels are decreased. Upon fasting, the normal elevation of peroxisome proliferator-activated receptor-alpha mRNA is lost in 11beta-HSD-1(-/-) mice, consistent with attenuated glucocorticoid induction. Despite this, crucial oxidative responses to fasting are maintained; carnitine palmitoyltransferase-I induction and glucose levels are similar to wild type. Refeeding shows exaggerated induction of genes encoding lipogenic enzymes and a more marked suppression of genes for fat catabolism in 11beta-HSD-1(-/-) mice, implying increased liver insulin sensitivity. Concordant with this, 24-h refed 11beta-HSD-1(-/-) mice have higher triglyceride but lower glucose levels. Further, 11beta-HSD-1(-/-) mice have improved glucose tolerance. These data suggest that 11beta-HSD-1 deficiency produces an improved lipid profile, hepatic insulin sensitization, and a potentially atheroprotective phenotype.
To study the role of alphaB-crystallin (alphaB) in the developing lens and its importance in lens structure and function.
Gene targeting in embryonic stem cells was used to generate mouse lines in which the alphaB gene and its protein product were absent. Gene structure and expression were characterized by genomic Southern blot, immunoblot, and Northern blot analyses, and two-dimensional gel electrophoresis. The gene knockout mice were screened for cataract with slit lamp biomicroscopy, and dissected lenses were examined with dark-field microscopy. Lenses and other tissues were analyzed by standard histology and immunohistochemistry. Chaperone activity was determined by heating lens homogenate supernatants and measuring absorbance changes.
In an unexpected result, lenses in the alphaB gene knockout mice developed normally and were remarkably similar to wild-type mouse lenses. All the other crystallins were present. The thermal stability of a lens homogenate supernatant was mildly compromised, and when oxidatively stressed in vivo with hyperbaric oxygen, the knockout lenses reacted similarly to wild type. In targeting the alphaB gene, the adjacent HSPB2 gene, which is not expressed in the lens, was also disrupted. Loss of alphaB and/or HSPB2 function leads to degeneration of some skeletal muscles.
AlphaB is not essential for normal development of a transparent lens in the mouse, and therefore is more dispensable to the lens than the closely related alphaA-crystallin. It may play a small role in maintaining transparency throughout life. alphaB and/or the closely related HSPB2 is required to maintain muscle cell integrity in some skeletal muscles.
Recent studies suggest that DNA polymerase eta (poleta) and DNA polymerase iota (poliota) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of poliota in generating mutations in an animal model, we first characterized the biochemical properties of murine poliota. Like its human counterpart, murine poliota is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols iota and eta. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of poliota. Analysis of hypermutation in variable genes from 129/SvJ (Poli-/-) and C57BL/6J (Poli+/+) mice revealed that the overall frequency and spectrum of mutation were normal in poliota-deficient mice. Thus, either poliota does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.
The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies.
The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp.
The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity.
The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.
Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.
A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.
Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
Cdt1 is required for loading the replicative DNA helicase MCM2/7, a process known as DNA replication licensing. Here we show that 129 mouse strains express a Cdt1 mutated allele with enhanced licensing activity. The mutation, named Δ(6)PEST, involves a six-amino acid deletion within a previously uncharacterized PEST-like domain. Cdt1 Δ(6)PEST and more extensive deletions exhibit increased re-replication and transformation activities that are independent of the Geminin and E3 ligase pathways. This PEST domain negatively regulates cell cycle-dependent chromatin recruitment of Cdt1 in G2/M phases of the cell cycle. Mass spectrometry analysis indicates that Cdt1 is phosphorylated at sites within the deleted PEST domain during mitosis. This study reveals a conserved new regulatory Cdt1 domain crucial for proper DNA licensing activity and suggests a mechanism by which the presence of Cdt1 in G2/M phases does not lead to premature origin licensing. These results also question the usage of 129 mouse strains for knockout analyses.
Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer
the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus,
we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells.
We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple
gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting
~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome
engineering.
Targeted mutagenesis in mice, a powerful tool for the analysis of gene function and human disease, makes extensive use of 129 mouse substrains. Although all are named 129, we document that outcrossing of these substrains, both deliberate and accidental, has lead to extensive genetic variability among substrains and embryonic stem cells derived from them. This clearer understanding of 129 substrain variability allows consideration of its negative impact on targeting technology, including: homologous recombination frequencies, preparation of inbred animals, and availability of appropriate controls. Based on these considerations we suggest a number of recommendations for future experimental design.
This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H2O2 in lens preparations. Rabbit (N/N1003A) and normal and GSHPx-1 transfected mouse (alpha TN4-1) lens epithelial cell lines and normal and GSHPx-1 transgenic and knockout mouse lenses were utilized. GSHPx-1 activity in the cell lines was increased from two-fold to about four-fold, in the lenses from transgenics more than four-fold and the lenses from knockouts had less than 3% of normal GSHPx-1 activity. The transgenic and knockout mice as well as their lenses appeared normal for up to 3 to 4 months, the longest period of observation. The preparations were subjected to oxidative stress by placing them either in a medium containing 120 or 300 microM H2O2 or utilizing photochemical stress where the H2O2 levels normally rise to about 100 microM over a few hours in the presence of a normal lens. With all preparations, it was found that either markedly increasing or eliminating GSHPx-1 activity had only a small effect on the system's ability to metabolize H2O2, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase (GSSG Red) and 3-aminotriazole (3-AT), an inhibitor of catalase, also had little effect. However, the addition of both inhibitors caused a marked decrease in H2O2 degradation. Examination of the distribution of GSHPx-1 in the lens indicated that the activity per milligram of protein was evenly distributed between the epithelium and the remainder of the lens in the normal lens and was about 1.7-fold greater in the epithelium of transgenic lenses than in the remainder of the lens. Surprisingly, the distribution of GSSG Red was quite different with eight- to ten-fold more activity in the epithelium. Catalase was also found to be concentrated in the epithelium. With H2O2 exposure, a rapid loss of non-protein thiol (NP-thiol) was found in cell cultures and in the epithelia of cultured lenses. However, the remainder of the lens showed little change in NP-thiol. The variation of GSHPx-1 activity did not influence the NP-thiol changes which occurred more rapidly and to a greater extent in the presence of BCNU. The addition of BCNU also caused a decrease in total lens NP-thiol. Examination of thymidine incorporation and choline transport, indicators of nuclear and membrane function, also reflects the H2O2 degradation data, showing little difference in the degree to which H2O2 effects these parameters in lenses from normal and transgenic animals. Catalase activity is four- to six-fold greater than GSHPX-1 activity in the alpha TN4-1 cell lines, about three-fold lower in the rabbit cell line and, remarkably, about 18-fold lower than the peroxidase in the normal mouse lens. In spite of such observations, the consistent overall conclusion is that GSHPx-1 and catalase function together but when GSHPx-1 is knocked out or GSSG Red is inhibited, catalase is able to protect the system from H2O2 stress. Indeed, the young mouse does not appear to require GSH Px-1 for normal function.
We report here the inactivation of a member of the Ice/Ced-3 (caspase) family of cell death genes, casp-11, by gene targeting. Like Ice-deficient mice, casp-11 mutant mice are resistant to endotoxic shock induced by lipopolysaccharide. Production of both IL-1alpha and IL-1beta after lipopolysaccharide stimulation, a crucial event during septic shock and an indication of ICE activation, is blocked in casp-11 mutant mice. casp-11 mutant embryonic fibroblast cells are resistant to apoptosis induced by overexpression of ICE. Furthermore, we found that pro-caspase-11 physically interacts with pro-ICE in cells, and the expression of casp-11 is essential for activation of ICE. Our data suggest that caspase-11 is a component of ICE complex and is required for the activation of ICE.
This communication investigates the effect of oxidative stress upon the lenses of young normal and glutathione peroxidase-1 (GSHPx-1) Knockout mice. Both normal and knockout lenses have similar biochemical and morpholigical characteristics and the elimination of GSHPx-1 only decreases slightly the ability of the lens to degrade H2O2. Examination of the effect of a 4 hr photochemical stress on morphological characteristics indicates that there is comparable damage in the normal and knockout lenses in the epithelial and bow regions while the posterior region remains normal. However, at 24 hrs post-insult, the normal lenses appear to recover somewhat in the bow region while the knockout bow and posterior regions have extensive damage. In contrast to the morphological data, the biochemical parameters (14C)choline transport and (3H)thymidine incorporation are affected to a somewhat greater extent in the knockout lenses than in normal lenses. While both of these parameters are further affected in the 24 hr post-insult period, there is no further change in the relative effects upon normal and knockout lenses. Non-protein thiol is affected in a similar manner in both lens types. The effect upon biochemical parameters of tertiary butyl hydroperoxide (TBHP) insult was similar to H2O2 and photochemical stress. The overall conclusion is that young GSHPx-1 knockout lenses handle oxidative stress somewhat less effectively than comparable normal lenses but non-stressed knockout lenses appear normal. These results differ from observations reported by Reddy et al. (1997) under somewhat different conditions.
NK cells are implicated in antiviral responses, bone marrow transplantation and tumor immunosurveillance. Their function is controlled, in part, through the Ly49 family of class I binding receptors. Inhibitory Ly49s suppress signaling, while activating Ly49s (i.e., Ly49D) activate NK cells via the DAP12 signaling chain. Activating Ly49 signaling has been studied primarily in C57BL/6 mice, however, 129 substrains are commonly used in gene-targeting experiments. In this study, we show that in contrast to C57BL/6 NK cells, cross-linking of DAP12-coupled receptors in 129/J mice induces phosphorylation of DAP12 but not calcium mobilization or cytokine production. Consistent with poor-activating Ly49 function, 129/J mice reject bone marrow less efficiently than C57BL/6 mice. Sequence analysis of receptors and DAP12 suggests no structural basis for inactivity, and both the 129/J and C57BL/6 receptors demonstrate normal function in a reconstituted receptor system. Most importantly, reconstitution of Ly49D in 129/J NK cells demonstrated that the signaling deficit is within the NK cells themselves. These unexpected findings bring into question any NK analysis of 129/J, 129Sv, or gene-targeted mice derived from these strains before complete backcrossing, and provide a possible explanation for the differences observed in the immune response of 129 mice in a variety of models.
Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.
Disrupted-In-Schizophrenia (DISC1) is a leading candidate schizophrenia susceptibility gene. Here, we describe a deletion variant in mDisc1 specific to the 129S6/SvEv strain that introduces a termination codon at exon 7, abolishes production of the full-length protein, and impairs working memory performance when transferred to the C57BL/6J genetic background. Our findings provide insights into how DISC1 variation contributes to schizophrenia susceptibility in humans and the behavioral divergence between 129S6/SvEv and C57BL/6J mouse strains and have implications for modeling psychiatric diseases in mice.
• animal model
• gene mutation
• psychiatric disorder
• schizophrenia
Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.
Cryopyrin is essential for caspase-1 activation triggered by Toll-like receptor (TLR) ligands in the presence of adenosine triphosphate (ATP). However, the events linking bacterial products and ATP to cryopyrin remain unclear. Here we demonstrate that cryopyrin-mediated caspase-1 activation proceeds independently of TLR signaling, thus dissociating caspase-1 activation and IL-1beta secretion. Instead, caspase-1 activation required pannexin-1, a hemichannel protein that interacts with the P2X(7) receptor. Direct cytosolic delivery of multiple bacterial products including lipopolysaccharide, but not flagellin, induced caspase-1 activation via cryopyrin in the absence of pannexin-1 activity or ATP stimulation. However, unlike Ipaf-dependent caspase-1 activation, stimulation of the pannexin-1-cryopyrin pathway by several intracellular bacteria was independent of a functional bacterial type III secretion system. These results provide evidence for cytosolic delivery and sensing of bacterial molecules as a unifying model for caspase-1 activation and position pannexin-1 as a mechanistic link between bacterial stimuli and the cryopyrin inflammasome.
Gene targeted or transgenic mice are frequently created on one genetic background and the mutation then transferred to a second genetic background by a series of backcrosses, with selection for the mutation at each generation. This is the same experimental design as that used in the production of classical congenic strains in which a genomic region from a “donor” strain is transferred to the background of a “recipient” strain by backcrossing and selection for ten or more generations. While the strategy largely results in the replacement of the donor background with the recipient background, the region flanking the selected gene remains of donor origin. Thus, the genes in this flanking region are “passengers” that travel with the selected gene, and when comparing transgenic (or knockout) mice with non-transgenic littermates, allelic differences in the passenger genes can conceivably influence the traits of interest. It now appears that this passenger gene effect may be an important confounder in studies of cardiovascular and metabolic traits that are influenced by dozens of common variations present among inbred mouse strains. Here, we discuss the problem and some approaches to avoid it.
Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype?
- R Gerlai
Gerlai, R. (1996). Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype? Trends Neurosci. 19, 177–181.
Disc1 is mutated in the 129S6/SvEv strain and modulates working memory in mice
- H Koike
- P A Arguello
- M Kvajo
- M Karayiorgou
- J A Gogos
Koike, H., Arguello, P.A., Kvajo, M., Karayiorgou, M., and Gogos, J.A. (2006).
Disc1 is mutated in the 129S6/SvEv strain and modulates working memory in
mice. Proc. Natl. Acad. Sci. USA 103, 3693-3697.