摘要
Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds.Generation of genetically edited pigs using somatic cell nuclear transfer(SCNT)or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder(FO).Herein,we developed a method termed germinal vesicle oocyte gene editing(GVGE)to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MII transition.Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential.The MII oocytes edited during in vitro maturation(IVM)could develop into blastocysts after parthenogenetic activation(PA)or in vitro fertilization(IVF).Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation.Up to81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos.In conclusion,GVGE might be a valuable method for the generation of non-mosaic maternal allele edited FO embryos in a short simple step.
Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds.Generation of genetically edited pigs using somatic cell nuclear transfer(SCNT)or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder(FO).Herein,we developed a method termed germinal vesicle oocyte gene editing(GVGE)to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MII transition.Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential.The MII oocytes edited during in vitro maturation(IVM)could develop into blastocysts after parthenogenetic activation(PA)or in vitro fertilization(IVF).Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation.Up to81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos.In conclusion,GVGE might be a valuable method for the generation of non-mosaic maternal allele edited FO embryos in a short simple step.
基金
supported by the National Key R&D Program of China(2017YFC1001901 and 2017YFA0102801)
the National Natural Science Foundation of China (31671540)
the National Transgenic Major Program (2016ZX08006003-006)
the Natural Science Foundation of Guangdong Province (2015A020212005 and 2014A030312011)
the Key R&D Program of Guangdong Province (2018B020203003)
the Guangzhou Science and Technology Project (201803010020)
