Ntages in high-throughput genetically modified animal production.Methods Ethics StatementThe Institutional Animal Care and Use Committee of The Jackson Laboratory approved all procedures used in this study and all mice were maintained at The Jackson Laboratory (Bar Harbor, ME, USA) in strict accordance with all institutional protocols and the Guide for the Care and Use of Laboratory Animals.Mouse Strains for PH Blastocyst Production and MicroinjectionTwo strains of mice were used to produce PH F1 blastocysts: B6;129-Gt(ROSA)26Sortm1(DTA)Mrc/J (JR010527), herein referred to as R26RDTA [15]; and FVB-Tg(Ddx4-cre)1Dcas/J (JR006954), herein referred to as Vasa-Cre [16]. Before use, the Vasa-Cre strain was backcrossed onto C57BL6/J (JR000664). Both strains were maintained with their transgenes in a homozygous state so that all F1 offspring would inherit the genes to undergo Cre directed stop codon excision and diphtheria toxin A expression in germ cells. To obtain early embryos, female R26RDTA mice were superovulated and mated with Vasa-Cre males. Reproductive tracts were collected and flushed at E2.5 dpc and harvested morula cultured overnight to blastocyst (E3.5 dpc). Conventional host blastocysts production used B6(Cg)-Tyrc-2J/J (JR000058) and embryos were obtained using the same methodology as above. For microinjection, 50?0 blastocysts per ESC clone were injected with 10?5 ESCs, depending upon embryo size. Approximately 11 blastocysts were implanted per pseudopregnant recipient CByB6F1/J female under isofluorane anesthesia, Carprofen was administered for post-surgical pain management. The same conditions were used for both conventional and PH blastocysts. Typically 50 of transferred blastocysts survived to term. At 7 weeks of age, all PH (putative chimeras) males were paired with C57BL/6NJ (ASP-015K custom synthesis JR005304) females. To determine the paternal genetic contribution of offspring derived from PH chimeras approximately 1 mm of tail or an ear punch was processed to crude DNA for genotyping. SNP genotyping was performed with a panel of 35 SNPs capable of unambiguously positively identifying the parental (germline) background of the ESC buy Gracillin versus the host embryo [17]. This included five SNPs which can distinguish between C57BL/6J (PH) and C57BL/6N ESC lines. IVF was performed with either freshly isolated sperm or sperm previously isolated from vasa deferentia and cryopreserved [18,19].HistologyFor sections, tissues were collected in Bouin’s Fixative, dehydrated in graded alcohols and then xylene. Tissues were paraffin-embedded, sectioned, deparaffinized, and stained with PAS (Periodic Acid Schiff) and photographed. The BtBr T+ Itpr3 tf/J (JR002282) and BALB/cJ (JR000651)derived ESC lines PB60.6 and PB150.18 ESC cells were isolated and provided by Predictive Biology, Inc. and are available through The Jackson Laboratory. The ESC line R1was derived from a 129X1/SvJ6129S1 cross [20]. The iPS cell line 9.48B (also known as 4.48B) was provided by the laboratory of Prof. Rudolf Jaenisch, and was derived from a cross of M26-M2rtTA6129sv; for complete lineage see [21]. Genetically modified IKMC C57BL/ 6N ESC lines were obtained from Eucomm, 23977191 CSD and through Regeneron Pharmaceuticals Inc. VelociGeneH program. ESC culture was conducted according to protocols provided by the ESC suppliers and as outlined in [22]. Briefly, CSD and EUCOMM ESC clones were grown in 10 FCS plus chemicalESC CultureImproved Germ Line of Embryonic Stem Cellsinhibitors. Regeneron Pharmaceuti.Ntages in high-throughput genetically modified animal production.Methods Ethics StatementThe Institutional Animal Care and Use Committee of The Jackson Laboratory approved all procedures used in this study and all mice were maintained at The Jackson Laboratory (Bar Harbor, ME, USA) in strict accordance with all institutional protocols and the Guide for the Care and Use of Laboratory Animals.Mouse Strains for PH Blastocyst Production and MicroinjectionTwo strains of mice were used to produce PH F1 blastocysts: B6;129-Gt(ROSA)26Sortm1(DTA)Mrc/J (JR010527), herein referred to as R26RDTA [15]; and FVB-Tg(Ddx4-cre)1Dcas/J (JR006954), herein referred to as Vasa-Cre [16]. Before use, the Vasa-Cre strain was backcrossed onto C57BL6/J (JR000664). Both strains were maintained with their transgenes in a homozygous state so that all F1 offspring would inherit the genes to undergo Cre directed stop codon excision and diphtheria toxin A expression in germ cells. To obtain early embryos, female R26RDTA mice were superovulated and mated with Vasa-Cre males. Reproductive tracts were collected and flushed at E2.5 dpc and harvested morula cultured overnight to blastocyst (E3.5 dpc). Conventional host blastocysts production used B6(Cg)-Tyrc-2J/J (JR000058) and embryos were obtained using the same methodology as above. For microinjection, 50?0 blastocysts per ESC clone were injected with 10?5 ESCs, depending upon embryo size. Approximately 11 blastocysts were implanted per pseudopregnant recipient CByB6F1/J female under isofluorane anesthesia, Carprofen was administered for post-surgical pain management. The same conditions were used for both conventional and PH blastocysts. Typically 50 of transferred blastocysts survived to term. At 7 weeks of age, all PH (putative chimeras) males were paired with C57BL/6NJ (JR005304) females. To determine the paternal genetic contribution of offspring derived from PH chimeras approximately 1 mm of tail or an ear punch was processed to crude DNA for genotyping. SNP genotyping was performed with a panel of 35 SNPs capable of unambiguously positively identifying the parental (germline) background of the ESC versus the host embryo [17]. This included five SNPs which can distinguish between C57BL/6J (PH) and C57BL/6N ESC lines. IVF was performed with either freshly isolated sperm or sperm previously isolated from vasa deferentia and cryopreserved [18,19].HistologyFor sections, tissues were collected in Bouin’s Fixative, dehydrated in graded alcohols and then xylene. Tissues were paraffin-embedded, sectioned, deparaffinized, and stained with PAS (Periodic Acid Schiff) and photographed. The BtBr T+ Itpr3 tf/J (JR002282) and BALB/cJ (JR000651)derived ESC lines PB60.6 and PB150.18 ESC cells were isolated and provided by Predictive Biology, Inc. and are available through The Jackson Laboratory. The ESC line R1was derived from a 129X1/SvJ6129S1 cross [20]. The iPS cell line 9.48B (also known as 4.48B) was provided by the laboratory of Prof. Rudolf Jaenisch, and was derived from a cross of M26-M2rtTA6129sv; for complete lineage see [21]. Genetically modified IKMC C57BL/ 6N ESC lines were obtained from Eucomm, 23977191 CSD and through Regeneron Pharmaceuticals Inc. VelociGeneH program. ESC culture was conducted according to protocols provided by the ESC suppliers and as outlined in [22]. Briefly, CSD and EUCOMM ESC clones were grown in 10 FCS plus chemicalESC CultureImproved Germ Line of Embryonic Stem Cellsinhibitors. Regeneron Pharmaceuti.
