CRISPR-Cas9介导的蒙古牛MSTN基因敲除细胞系的建立

doi:10.16431/j.cnki.1671-7236.2018.04.001

Abstract

Abstract:

This study was aimed to edit myostatin (MSTN) gene of Mongolian bovine fetal fibroblasts using CRISPR-Cas9 system,build an unmarked Mongolian bovine cell line which its MSTN gene had been knocked out,and provide experimental material for cultivating muscular Mongolian bovine.Skin fibroblast cell lines were established on 45 day of gestation in Mongolia bovine fetus by tissue adherent method,at the same time,a gRNA target site in exon 2 region was selected,and three high scores sgRNAs sequences which designed in online software were chosen,unmarked Cas9/gRNA vector was constructed by kit method,its activities was tested by T7E1 digestion,the active vector was transfected into Mongolian bovine fetal fibroblasts by electric transfer,and seed transfected single cells in 96-well plates by infinite dilution method and pipette method,which DNA was extracted,and obtained positive cells by PCR amplification and sequencing verification in the end.The results showed that we constructed three unmarked CRISPR-Cas9 vectors and found that only one vector was active.A total of 96 single cell strain were obtained by screening single cells which after electrical transfection,and only one single cell strain was found that occur single-base mutation after sequencing analysis,which made MSTN gene could not encode normal protein.This study established successfully a MSTN gene inactivated cell line,which could be used as a donor cell for subsequent somatic cell nuclear transfer for the production of unmarked Mongolian bovine with MSTN gene knockouted.It was of great significance to cultivate Mongolian bovine with high meat production rate and biosecurity.

Key words: CRISPR-Cas9 system; MSTN gene; Mongolia bovine; non-lable cell line

CLC Number:

Q812

GUO Ziru, ZHANG Li, MA Yunlong, SU Xiaohu, ZHOU Huanmin, ZHANG Yanru, LIU Xiao. Establishment of MSTN Gene Knockout in Mongolia Bovine Cell Lines Mediated by CRISPR-Cas9 System[J]. , 2018, 45(4): 841-849.

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Establishment of MSTN Gene Knockout in Mongolia Bovine Cell Lines Mediated by CRISPR-Cas9 System

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