CRISPR/Cas9介导的绵羊胎儿成纤维细胞肌肉生长抑制素基因敲除研究

doi:10.16431/j.cnki.1671-7236.2015.11.001

Abstract

Abstract: Mutation in myostatin (MSTN) gene resulted in double muscle effect,generating more mutton.To knock out MSTN gene in sheep fetal fibroblast by CRISPR/Cas9 system and obtain MSTN gene knockout cell lines,four plasmids were designed and constructed to target MSTN gene,and confirmed correctly by sequencing.The correct plasmids were delivered into the fetal fibroblast cells.The targeting efficiency was detected using SURVEYOR assay Kit.The stable transfected cell colonies were obtained via limiting dilution procedure.The sequence results demonstrated that the pX330-target 1 and pX330-target 4 plasmids could successfully knockout MSTN gene,and the targeting efficiency were 24.20% and 10.18%,respectively.Twelve MSTN gene knockout cell colonies were obtained via limiting dilution,and one of them was homozygous mutation.Several indel mutations were discovered at specific site,and some of them were frame-shift mutation.Therefore,we concluded that the CRISPR/Cas9 system could apply to the gene editing of sheep efficiently,and the gene knockout cell clones had potential application in generating MSTN gene knockout sheep.

Key words: CRISPR/Cas9 system; myostatin; gene knockout; sheep

CLC Number:

LI Cong, CAO Wen-guang. Knockout of Myostatin Gene by CRISPR/Cas9 System in Ovine Fetal Fibroblasts[J]. , 2015, 42(11): 2813-2821.

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Knockout of Myostatin Gene by CRISPR/Cas9 System in Ovine Fetal Fibroblasts

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