Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line. Mamm Genome

Transgenic Animal Model Core, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Mammalian Genome (Impact Factor: 3.07). 09/2007; 18(8):549-58. DOI: 10.1007/s00335-007-9054-0
Source: PubMed


Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background.

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Available from: Christopher D Pacheco, Jul 08, 2014
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    • "Homozygous mice carrying the 1d2d-PSD-95-EGFP gene were obtained by mating heterozygous littermates and analyzed by polymerase chain reaction (PCR) genotyping using primers for the exon 4 antisense strand and exon 8 sense strand (Figure  1D). The Bruce4 C57BL/6 J ES cell DNA sample varies at 34 markers (12.4%) from C57BL/6 J DNA among 275 simple sequence length polymorphisms tested [40]. DNA from homozygous 1d2d-PSD-95-EGFP or littermate wild-type (WT) mice showed variations at 2.5 of those 34 markers in an average of 2 mice. "
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    ABSTRACT: Background Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. Results The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. Conclusions These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.
    Molecular Brain 12/2012; 5(1):43. DOI:10.1186/1756-6606-5-43 · 4.90 Impact Factor
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    • "C57BL/6 and 129 ES cell lines were maintained in high glucose Dulbecco's minimal essential medium (Invitrogen) supplemented with 15% (v/v) fetal bovine serum (Harlan), 4 mM glutamine, 1 μM 2-mercaptoethanol, 1% MEM non-essential amino acids, 50 IU/ml penicillin, 50 μg/ml streptomycin and 1000 IU/ml ESGRO (Millipore) on FVB/N mouse embryonic feeder cells mitotically inactivated by irradiation. Before DNA extraction and genotyping, ES cell lines were passaged twice on gelatin-coated dishes to eliminate feeders from the cultures as described in [15]. "
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    ABSTRACT: 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.
    Biochemical Journal 02/2012; 443(2):355-9. DOI:10.1042/BJ20120249 · 4.40 Impact Factor
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    • "Blastocysts were isolated from inter-crossing of Sag +/− mice or Sag +/− ;Nf1 +/− mice, and placed in culture on irradiated mouse embryonic feeder cells in high glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with 15% FBS (Harlan, Indianopolis, IN), 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO), 10 3 U/ml LIF (ESGRO, Millipore),25 μM PD098059 (Sigma) and penicillin/streptomycin. Inner cell mass outgrowths were trypsinized and passaged until ES cell lines were established in 35 mm cell culture dishes (Hughes et al., 2007). "
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    ABSTRACT: SAG/RBX2/ROC2 protein is an essential RING component of SCF E3 ubiquitin ligase. The role of SAG during embryogenesis remains unknown. We report a critical role for SAG in controlling vascular and neural development by modulating RAS activity via promoting degradation of neurofibromatosis type 1 (NF1). Mice mutant for Sag died at embryonic day 11.5-12.5 with severe abnormalities in vascular and nervous system. Sag inactivation caused Nf1 accumulation and Ras inhibition, which blocks embryonic stem (ES) cells from undergoing endothelial differentiation and inhibits angiogenesis and proliferation in teratomas. Simultaneous Nf1 deletion fully rescues the differentiation defects in Sag(-/-) ES cells and partially rescues vascular and neural defects in Sag(-/-) embryos, suggesting that the effects of Sag deletion may not be solely explained by Nf1 misregulation. Collectively, our study identifies NF1 as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase and establishes a ubiquitin-dependent regulatory mechanism for the NF1-RAS pathway during embryogenesis.
    Developmental Cell 11/2011; 21(6):1062-76. DOI:10.1016/j.devcel.2011.09.014 · 9.71 Impact Factor
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