用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因

doi:10.6048/j.issn.1001-4330.2021.09.022

新疆农业科学 ›› 2021, Vol. 58 ›› Issue (9): 1747-1755.DOI: 10.6048/j.issn.1001-4330.2021.09.022

• 园艺特产·微生物·畜牧兽医·农业经济 • 上一篇下一篇

用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因

郭延华, 皮文辉

绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所,新疆石河子 832000

收稿日期:2020-07-13 出版日期:2021-09-20 发布日期:2021-09-29
通信作者: 皮文辉(1972-),男,新疆人,研究员,博士,研究方向为动物遗传育种与繁殖,(E-mail)wzjpwh＠163.com
作者简介:郭延华(1983-),男,新疆人,助理研究员,硕士,研究方向为动物遗传育种与繁殖,(E-mail)282437680@qq.com
基金资助:
国家自然科学基金(31560321)

Use of the CRISPR-Cas9 System in Sheep Fibroblasts to Lead Fluorescent Tags into ACTG1 Gene

GUO Yanhua, PI Wenhui

State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi Xinjiang 832000, China

Received:2020-07-13 Online:2021-09-20 Published:2021-09-29
Correspondence author: PI Wenhui (1972-), male, Researcher, research field: animal genetic breeding and reproduction, (E-mail) wzjpwh@163.com
Supported by:
Supported by the Project of the National Natural Science Foundation of China (31560321)

摘要/Abstract

摘要： 【目的】在绵羊成纤维细胞中,针对ACTG1基因羧基端,定点导入荧光蛋白标记基因,将外源基因定点导入绵羊基因组中,建立有效的方法。【方法】CRISPR-Cas9系统在绵羊成纤维细胞基因组特定区域引起DNA双链断裂,从而诱导细胞修复断裂的的基因组。通过NHEJ修复途径,在特定位点导入外源基因,改善定点导入效率。【结果】采用CRISPaint通用供体模板,结合CRISPR-Cas9系统,在绵羊成纤维细胞ACTG1基因导入荧光标记效率达1.4%,获得了外源基因定点导入的单克隆细胞株。【结论】CRISPR-Cas9系统结合NHEJ,能够有效的将大的外源DNA序列导入绵羊成纤维细胞的预定基因组位点。

关键词: 绵羊; 成纤维细胞; ACTG1; CRISPR-Cas9

Abstract: 【Objective】 This project aims to establish a method for leading foreign genes into the specific site of sheep fibroblast genome, hoping applying this method allows creating C-terminal tag fusions of endogenously encoded proteins in sheep ACTG1 gene with high efficiencies.【Method】 The CRISPR-Cas9 system caused double-strand to break in specific regions of cells, thus inducing cells to repair broken genomes, and the non-homologous end joining (NHEJ) repair pathway was used to improve knock-in efficiency of the exogenous gene.【Result】 The fluorescent protein open reading frame (ORF) was inserted into ACTG1 gene of sheep fibroblasts by the CRISPaint universal donor and CRISPR-Cas9 system. The insertion efficiency of exogenous gene achieved 1.4% of the total cells. Monoclonal cell lines were obtained for site-specific introduction of foreign genes.【Conclusion】 This technique holds promise for quick and efficient insertion of a large foreign DNA sequence into a predetermined genomic site in sheep fibroblasts by CRISPR-Cas9 system and NHEJ repair pathway.

Key words: sheep; fibroblasts; ACTG1; CRISPR-Cas9

中图分类号:

S852.6
S826

郭延华, 皮文辉. 用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因[J]. 新疆农业科学, 2021, 58(9): 1747-1755.

GUO Yanhua, PI Wenhui. Use of the CRISPR-Cas9 System in Sheep Fibroblasts to Lead Fluorescent Tags into ACTG1 Gene[J]. Xinjiang Agricultural Sciences, 2021, 58(9): 1747-1755.

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用CRISPR-Cas9在绵羊成纤维细胞ACTG1导入荧光标记基因

Use of the CRISPR-Cas9 System in Sheep Fibroblasts to Lead Fluorescent Tags into ACTG1 Gene

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