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Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes.
Journal of Genetics and Genomics ( IF 5.9 ) Pub Date : 2019-07-20 , DOI: 10.1016/j.jgg.2019.07.002
Xiaohu Su 1 , Wei Chen 2 , Qingqing Cai 2 , Puping Liang 1 , Yaosheng Chen 2 , Peiqing Cong 2 , Junjiu Huang 3
Affiliation  

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 (F0). 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 to 81.3% 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 F0 embryos in a short simple step.



中文翻译:

通过将CRISPR / Cas9注射到生小泡卵母细胞中来生产非镶嵌基因组编辑的猪胚胎。

转基因猪代表了产生人类疾病模型和生产新品种的巨大希望。由于创建者(F0)的低发育率或镶嵌性,使用体细胞核移植(SCNT)或合子细胞质显微注射生成基因编辑的猪是一个繁琐的过程。本文中,我们开发了一种称为生胚囊卵母细胞基因编辑(GVGE)的方法,可通过在GV到MⅡ的过渡过程中编辑母本等位基因来生产非镶嵌性猪胚胎。向GV卵母细胞注射Cas9 mRNA和X连锁的Dmd基因特异性gRNA不会影响它们的发育潜能。体外成熟(IVM)过程编辑的MⅡ卵母细胞可能在孤雌激活(PA)或体外受精(IVF)。基因分型结果表明,在卵母细胞成熟过程中,可以有效地编辑与母体基因X连锁的Dmd。高达81.3%的已编辑IVF胚胎是非马赛克Dmd基因突变体胚胎。总之,GVGE可能是在短短的简单步骤中生成非马赛克母亲等位基因编辑的F0胚胎的有价值的方法。

更新日期:2019-07-20
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