[Show abstract][Hide abstract] ABSTRACT: Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.
[Show abstract][Hide abstract] ABSTRACT: The Sumatran rhinoceros (Dicerorhinus sumatrensis) is on the verge of extinction. Once found throughout Southeast Asia, it stands now with less than 100 individuals scattered mainly in three national parks in Sumatra. In Sabah (Malaysian Borneo), as well as in Peninsular Malaysia, the Sumatran rhinoceros is now considered to be " functionally extinct ". Between 1984 and 2014 forty-five Sumatran rhinoceroses were captured from the wild, resulting in only two successful breeding pairs. In early 2015 only nine individuals survive in captivity: three (1.2) in the Borneo Rhino Sanctuary, in Sabah; five (2.3) in the Sumatran Rhino Sanctuary, in Sumatra; and only one (1.0) left in Cincinnati Zoo, USA. Since 2009 the Leibniz Institute for Zoo and Wildlife Research (IZW) has been collaborating with the Borneo Rhino Alliance (BORA), mainly through the use of advanced imaging and assisted reproduction technologies on the 1.3 wild-caught Bornean rhinoceroses (Dicerorhinus sumatrensis harrissoni) held at the Borneo Rhino Sanctuary. Throughout fourteen visits to Sabah, the IZW team and associated experts had the chance to perform forty-two reproductive assessments, six semen collections by electroejaculation, three endometrial cyst removal procedures, one artificial insemination (AI), three oocyte collections (" ovum pickup " , OPU) and one intracytoplasmic sperm injection (ICSI). In what concerns the male, significant improvements in semen quality were observed throughout time: despite being considered reproductively inactive in 2009 and 2011, in 2014 a maximum concentration of 5x10 6 sperm cells /mL was obtained, with 65 % motility. When captured from the wild, two cycling females showed severe reproductive pathology that rendered them incapable of carrying a pregnancy, namely extensive cystic endometrial hyperplasia and a large number of uterine leiomyomas. Four different techniques where used to remove endometrial cysts: uterine lavage with cell medium M199 (Sigma-Aldrich Chemie GmbH, Munich, Germany) and povidone-iodine solution (Braunol®, B. Braun Melsungen AG, Germany), endoscopic assisted laser photoablation and ultrasound guided aspiration. These procedures proved to be of very limited success. Consequently, AI was attempted only once and no fertilisation occurred due to poor condition of the uterus and irregular cycling. As natural conception was excluded, in vitro fertilisation was attempted for the first time in Sumatran rhinoceros. OPU was performed on three occasions and successful on two, with the total collection of five oocytes. ICSI was performed with two of the oocytes, but cleavage was not achieved. Furthermore, to preserve the genome of these that may well be the last Bornean rhinoceroses, cell cultures were established from skin and mucosal samples and cryopreserved until seven passages, for future development of induced pluripotent stem cells.
Int Conf Dis Zoo Wild Anim 2015, Barcelona, Spain; 05/2015
[Show abstract][Hide abstract] ABSTRACT: Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (TFAM), mtDNA polymerase ? subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (PP=0.01) and lower mtDNA:total DNA ratios (Pin vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.
Reproduction Fertility and Development 04/2015; 27(6). DOI:10.1071/RD14450 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracytoplasmic sperm injection (ICSI) is an established method to fertilise equine oocytes, but not all oocytes cleave after ICSI. The aims of the present study were to examine cytoskeleton patterns in oocytes after aging in vitro for 0, 24 or 48h (Experiment 1) and in potential zygotes that failed to cleave after ICSI of oocytes from donors of different ages (Experiment 2). Cytoplasmic multiasters were observed after oocyte aging for 48h (P<0.01). A similar increase in multiasters was observed with an increased interval after ICSI for young mares (9-13 years) but not old (20-25 years) mares. Actin vesicles were observed more frequently in sperm-injected oocytes from old than young mares. In the present study, multiasters appeared to be associated with cell aging, whereas actin vesicles were associated with aging of the oocyte donor.
Reproduction Fertility and Development 03/2015; 27(6). DOI:10.1071/RD14468 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3ngmL(-1) nocodazole for 3h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5μgmL(-1) cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3kVcm(-1) DC for 30μs in 0.3M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1mM SrCl2 in Ca(2+)-free KSOM medium for 2 to 2.5 or 5 to 6h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments=15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n=19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n=623 v. 100%, n=80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P=0.07), and their ability to implant (52.6% v. 38.2%; P=0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n=5; P=0.08), and produced live fetuses (4.9%, n=3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.
Reproduction Fertility and Development 12/2014; 27(1):113. DOI:10.1071/RDv27n1Ab41 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracytoplasmic sperm injection (ICSI) is used for assisted fertilization of equine oocytes. However, not all oocytes cleave after ICSI. Maternal aging deleteriously affects fertility in mares and women, with reduced oocyte quality and success of assisted reproductive technologies. In the oocyte, senescence and cell-programmed death begins after maturation; the extent that maternal age affects these events is unknown. We hypothesised that formation of α/β tubulin asters and f-actin bubbles are associated with aging of the oocyte in vitro and/or aging of the oocyte in vivo, in aged donors. In Exp 1, oocytes were collected from ovaries obtained from an abattoir and matured for 28h and selected for polar body extrusion (0h). At 0, 24, and 48h, oocytes (n=38 total) were fixed in MTSB-XF and transferred into wash solution with 1% BSA and 0.1% Triton X-100 in PBS for immunostaining. For experiment 2, oocytes were collected from preovulatory follicles of mares (9-25 yr) in a clinical ICSI program and injected with sperm from various stallions after extrusion of a polar body. Between 24 to 51h after ICSI, uncleaved oocytes (n=52, single cell without evidence of fragmentation or indentation of the oolemma) were fixed. All oocytes were incubated with α/β tubulin and human-anti-centromere antibody-CREST/ACA (1:100 each). Following primary incubation, oocytes were washed and incubated with Alexa 488, Alexa 647, Alexa 561-phalloidin, and Hoechst 33258. Images and Z-stacks were acquired on an Olympus IX81 spinning disk confocal microscope. Morphometric and intensity analyses of images were performed using SlideBook software (Denver, CO). Student's t-test, Fisher's exact test, and chi-square analyses were used for statistical comparisons. After aging in vitro (experiment 1), the number of oocytes with tubulin multiasters increased (P<0.001; 9% at 0h, 14% at 24h, 85% at 48h); however, actin bubbling was observed in only 5/38 (13%) oocytes, with no effect of incubation time. In experiment 2, tubulin multiasters were present in 62% of oocytes that failed to cleave. More multiasters were observed per oocyte from mares ≤13 yr than ≥20 yr (P=0.03) and fixed at 24 to 28h than 44 to 51h (P=0.04). Actin bubbles were observed in 71% of oocytes that failed to cleave after ICSI, with more actin bubbles in oocytes from mares ≥20 yr than ≤13 yr (P=0.01) and fixed 44 to 51h versus 24 to 28h after ICSI (P=0.05). The sum intensity and area of the actin bubbles were higher in oocytes fixed at 44 to 51h than 24 to 28h (P=0.01 and P=0.04). The area occupied by the actin bubbles was larger (P=0.05) in oocytes from mares ≥20 yr than ≤13 yr. This study demonstrates actin bubbles and tubulin asters are involved in oocyte aging and cytoskeleton remodelling with or without fertilization. Although actin structures were associated with donor age and hours after ICSI, they were not present in unfertilized oocytes aged in vitro. Multiaster formation was associated with cell senescence in oocytes aged in vitro. Although not previously reported for the equine oocyte, multiaster formation appeared to be an initial fertilization event within the oocyte associated with attempted zygote development.
Reproduction Fertility and Development 12/2014; 27(1):249-50. DOI:10.1071/RDv27n1Ab321 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this article was to compare plasma estrone sulfate (E1SO4), clinical biochemistry, and milk yield of dairy cows carrying a female fetus from a bull (BULL) or from its clone (CLONE), evaluating also the relationship between the former variables and the birth weight of the newborn. Sixteen recipient dairy Friesian heifers (10 BULL and 7 CLONE) received a female embryo, obtained by in vitro embryo production and sexing by polymerase chain reaction with the semen of the BULL or the CLONE. Blood samples on all cows were obtained before feed distribution in the morning from jugular vein from 4 weeks before to 4 weeks after calving, to be analyzed for metabolic profile. The samples from late gestation were also analyzed for E1SO4 concentration. To separately assess the effect of calf birth weight (CBW), data were categorized as follows: low (<39 kg; BWT-A), mid (39-46 kg; BWT-B), and high (>46 kg; BWT-C). The plasma concentrations of β-hydroxybutyric acid (BHB, P = 0.019), Na (P = 0.002), Cl (P = 0.026), strong cation-anion balance (P = 0.020), total bilirubin (P = 0.054), and α1-globulin (P = 0.044) were higher in prepartum BULL recipients than those in CLONE, whereas BHB (P = 0.021) and Mg (P = 0.090) were higher in postpartum BULL recipients, while no differences were recorded in the remaining postpartum parameters. The CBW class had significant interaction with week of gestation on antepartum plasma estrone sulfate (P = 0.021), whereas CBW per se affected antepartum plasma BHB (P = 0.021), and nonesterified fatty acids (NEFA; P = 0.011) being higher in BWT-C which also had the lower NEFA concentration during postpartum. Milk yield was unaffected by the sire used, both for quantitative and qualitative aspects. Cows carrying heavier fetus (BWT-C) had a different lactation affected by month compared with the other 2 CBW groups. From these results, there were no differences between BULL and CLONE recipients. Estrone sulfate, BHB, and NEFA may be used to predict CBW and provide different nutritional management during gestation.
[Show abstract][Hide abstract] ABSTRACT: A number of in vitro toxicity assays based on human embryonic stem cells (hESCs) are under development in order to provide alternative methods for the screening of chemicals and drugs and to reduce the number of animals needed for developmental toxicity assessment. The major challenge is to demonstrate the reliability of these in vitro methods by correlating the in vitro produced results to the available in vivo data. In this context transcriptomic approaches associated to toxicogenomic database analysis give the possibility to screen, annotate and cluster high numbers of genes and to identify the molecular changes that univocally mark the toxicity induced processes or are indicative of the early initiating events that lead to cellular toxicity. In this retrospective study we compare microarray transcriptomic data derived from two different hESCs lines (HUES1 and H9) exposed to valproic acid (VA) while applying the same differentiation protocol. We present the results of this comparative analysis in light of the known teratogenic effects of VA. The results show molecular changes in the processes of neural development, neural crest migration, apoptosis and regulation of transcription, indicating a good correspondence with the available in vivo data. We also describe common toxicological signatures and provide an interpretation of the observed qualitative differences referring to known biological features of the two hESCs lines.
[Show abstract][Hide abstract] ABSTRACT: Background
Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors.Methods
In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model.ResultsCorneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals.Conclusions
Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.
[Show abstract][Hide abstract] ABSTRACT: Amniotic fluid (AF) is a source of multipotent mesenchymal stem cells (MSCs), very promising cells for tissue engineering in clinical application. The aim of this work was to isolate and characterize cells isolated from bovine AF as alternative sources of primitive multipotent stem cells in a species that could be a large-animal model for biomedical and biotechnology researches. Samples were recovered, at slaughterhouse, from 39 pregnant cows at different trimesters of pregnancy and cells were cultured in vitro. At passages (P) 3 and 7 differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced. Flow cytometry analysis for CD90, CD105, CD73, CD44, CD34, CD45 and CD14 was performed, immunocytochemistry (ICC) for Oct4, SSEA4, α-SMA, Vimentin, N- and E- Cadherin and CK and qPCR analysis for OCT4, NANOG and SOX2 were carried out. The cell yield was significantly higher in the first trimester compared to the second and the third one (P < 0.05). Cells were isolated from 25/39 samples and cell population appeared heterogeneous. Two main cell types were identified in samples from all trimesters: round- (RS) and spindle-shaped (SS) cells. 17/25 samples showed both populations (mixed, MX). Both cell types showed MSC-markers and differentiation capability with some variability related to the passages. The SS-population also expressed low levels of stemness markers such as NANOG and SSEA4 but not OCT4. Bovine AF shows a heterogeneous cell population containing also MSCs, multipotent cells that represent an intermediate stage between embryonic stem cells and adult ones.
[Show abstract][Hide abstract] ABSTRACT: Objective: To investigate the telomere length in bovine offspring produced by a cloned and control bull, and the telomerase activity in embryos produced with the same technology. Methods: Five daughters of a control and five daughters of a bull cloned using a fibroblast of the control were produced by IVF using sperm of the two bulls. Blood samples of the offspring were collected at 2, 6, and 12 months of age and the relative telomere length (RTL) was assessed by flow cytometry. At same time the body growth, hematological profile, and clinical biochemistry of the same progeny was extensively surveyed, and results have been reported in a previous work. Thereafter, the telomerase activity was assessed using a real time PCR quantitative assay in groups of embryos produced with the same technology. Results: The offspring of the clone exhibited a modest, but significant (P<0.05), shortening of the telomeres (21.36%, 20.56% and 20.56%) compared to that of the CONTROL (23.78%, 23.53% and 22.43%) as mean values determined at 2, 6 and 12 months, respectively. Shortening of telomeres in respect to the age was not significant. No statistical difference was reported between telomerase activity assessed in 144 cloned (3.4-03 ± 2.4-03 amoles/µL) and 80 control (2.1-03 ± 1.8-03 amoles/µL) embryos. Conclusions: The results have revealed a moderate shortening of telomeres in the offspring of the clone with respect to control. However, this study did not evidence differences in the two progenies that suggest welfare problems during the first year of life.
[Show abstract][Hide abstract] ABSTRACT: The physiology of the reproductive cycle is extremely complex due to the several cell types, somatic and germ cells, that are present in the different organs and due to the finely regulated mechanisms that interact not only within reproductive tissues but also in concert with an upper regulatory and feedback control system at the level of the central nervous system. During lifetime, reproductive physiology changes dramatically, both in male and in female sex, passing from a relatively inert prepuberal stage to hormonally driven puberty entering the fertile period of life. Regular female reproductive cyclicity is interrupted by pregnancy establishment and by the development of the conceptus that become the most sensitive targets for potential environmental insults and second-generation effects. Therefore the design of relevant and reliable alternative tests capable of measuring and predicting adverse effects on the multitude of reproductive functions in the different cells/organs is an enormous task technically and scientifically. This chapter provides an overview of the available alternative tests for reproductive toxicity, in relation to the present regulatory framework and with more emphasis on those tests and results that have been developed and obtained within international research initiatives. Tests specifically designed for detecting endocrine effects are described in more detail in another chapter of this book.
[Show abstract][Hide abstract] ABSTRACT: Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.
[Show abstract][Hide abstract] ABSTRACT: In recent years, fetal adnexa and fluids have been recognised as important sources of mesenchymal stem cells (MSC). The aim of this study was to characterise cell populations of bovine amniotic fluid, studying phenotypic characterisation, RNA expression, and differentiation potential of samples after in vitro culture for different lengths of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at the slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1:1) plus 10% fetal bovine serum (FBS) in 5% CO2 at 38.5°C. At passages P3 and P7, a sample for each of the 4 population found was characterised. Immunophenotypic characterisation was performed for MSC (CD90, CD105, CD44) and haematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4, and α-SMA and the ratio between positive cells and total nuclei was evaluated. Gene expression profile was analysed by RT-PCR for pluripotency markers (Oct4, Nanog, Sox2). At the same passages chondrogenic, osteogenic and adipogenic differentiation were induced and evaluated morphologically and cytologically using, respectively, Alcian blue to identify cartilage matrix, Von Kossa for extracellular calcium deposition, and Oil Red O for intracellular lipid droplets. Cell population appeared heterogeneous and we could identify 2 main cell types: round (R) and spindle-shaped (S) cells. Each isolated sample was classified into one of the following 4 types depending on percentages of R or S cells: prevalence of S-cells (S), prevalence of R-cells (R), and samples showing both morphologies with ~10% of S-cells (S10) or 40% S-cells (S40). S-cells percentage decreased with passages in S10 and S40. After FACS, all lines were positive for CD90, CD105, CD44, and CD34 and negative for CD14 both at P3 and at P7. After ICC, Oct4 was negative in all samples analysed, few S cells stained for SSEA4 (8%) at P3 but increased at P7 to 22%; R, S10, and S40 did not express SSEA4 both at P3 and at P7. α-SMA was expressed in all samples at P3 (9.4% S; 0.9% R; 2.5% S10; 27% S40) but not at P7 (27.5% S; 0% R; 0% S10; 0% S40). After RT-PCR analyses, Oct4 was negative in all samples; at P3, Nanog was clearly positive in S-cells, weak in S40, and negative in R and S10, but all samples turned negative at P7. Sox2 was weakly expressed (S) or not expressed (S10, S40, R) at P3 and it was negative in all cells at P7. Only S showed high differentiation potential into all 3 lineages at both P3 and P7, R had the lowest differentiation potential, whereas S10 and S40 were intermediate at both end points. In conclusion, bovine amniotic fluid showed heterogeneous cell populations and S-type had the characteristics of MSCs. S10 and S40 showed more MSC markers at P3, when S cells were still present, and this aspect suggests that S population is the presumptive MSC one. Although prevalent, R-type showed only some MSC characteristics. Further studies are under way to improve S-type isolation, purification, and culture, and to determine the lifespan of these cell types.
Reproduction Fertility and Development 12/2013; 26(1):207-8. DOI:10.1071/RDv26n1Ab186 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, site-specific nucleases (zinc-finger nucleases, ZFN; TAL effector nucleases; and CRISPR) emerged as powerful tools for gene modification of different cells types and enhanced green fluorescent protein (EGFP)-specific ZFN were successfully used in the rat (Geurtz et al. 2010) and in the pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008 Clon. Stem Cells), we generated an EGFP transgenic porcine line (Verro2GFP) characterised by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission, and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated recombinase-mediated cassette exchange (RMCE), using EGFP-specific ZFN. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment=left-homology-arm=LHA; polyA sequence=right-homology-arm=RHA). Cloning floxed (lox2272/lox5171) hygromycin resistance coding sequence between LHA and RHA sequences, we generated the targeting/RMCE vector (pB5'3'Hygro-PL) and its positive control (C+) for PCR set-up (100-1000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM+M199(1:1)+10% FCS, bFGF in 5% CO2, 5% O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2μg of each ZFN coding vector (Sigma-CompoZr(®)) and 2μg of pB5'3'Hygro-PL/KpnI vector were used to "nucleofect" 1.4×10(6) Verro2GFP fibroblasts in 2 experiments. Transfected cells were plated in 20 Petri dishes (Ø=150mm) and cultured under hygromycin selection (200μgmL(-1)) for 15 days. After 12 days of drug selection, 82 resistant colonies were picked up and expanded in 24 multiwell plates for SCNT. All colonies were PCR screened and 45 (54.9%) colonies were positive. Four colonies were used in zona-free SCNT experiments with 140 Day 6 compacted morulae/blastocysts transferred into 2 synchronized sows that both became pregnant. One pregnancy went to term and delivered 5 live animals and 5 stillborn with correct hygromycin cassette integration, detected by PCR. The PCR products were sequenced in 7 animals to verify integration of promoterless targeting vector and in all 7 sequenced samples we obtained a correct insertion without any substitution/deletion. Using hygromycin selection in these experiments, we demonstrated that ZFN-mediated gene targeting can be easily done with high efficiency and is compatible with living animals. Moreover, we have validated a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications.
Reproduction Fertility and Development 12/2013; 26(1):221-2. DOI:10.1071/RDv26n1Ab215 · 2.40 Impact Factor