利用TALENs技术建立基因定点修饰的猪Oct-4-EGFP细胞系  

Estblishment of Porcine Oct-4-EGFP Site-directed Transgene Cell Lines using Engineered TALENs

作　　者：刘畅[1] 冯冲[1] 宋志强[1,2] 李西睿[1] 王宁[1] 储明星[1] 潘登科[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,农业部畜禽遗传资源与种质创新重点实验室,北京100193 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国畜牧兽医》2013年第11期1-6,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金面上项目(30972095);中央级公益性科研院所基本科研业务费项目(2011cj-10-2)

摘　　要：本研究旨在建立一个用于干细胞示踪的报告体系,将EGFP cDNA及两侧带有LoxP位点的neo抗性基因插入五指山小型猪内源性Oct-4基因的终止密码子处,以Oct-4基因完整的5′调控区启动EGFP的表达,为猪干细胞研究提供有价值的工具。试验定制了针对猪Oct-4终止密码子的TALENs,与打靶载体共转染猪耳成纤维细胞,药物筛选得到抗性克隆点514个,经过PCR鉴定,共获得杂合阳性克隆点36个,打靶效率分别为5.6%和13.0%。本研究成功获得了Oct-4-EGFP转基因细胞系,并证明了TALENs技术可以明显提高同源重组效率。This study aims to establish a reporting system for stem cells tracer, EGFP cDNA together with LoxP-flanked PGK-neo cassettes were inseet into the Oct-4 gene of Wuzhishan Miniature pig at the stop codon via TALENs mediated homolo- gous recombination, expression of EGFP gene was under control of Oct-4 5＇-regulatory region. TALENs directed against se- quences flanking the stop eodon of Oct 4 were cotrasfected with targeting vector, 514 drug-resistant cell clones were screened, and 36 correctly targeted clones were confirmed by PCR from them, the targeting efficiency were 5.6% and 13.0%. Oct-4- EGFP transgenic cell line was establinshed in this study, and it was also proved that TALENs could significantly improve the efficiency of homologous recombination.

关 键 词：Oct-4-EGFP TALENs 同源重组 猪 干细胞 多能性 

分 类 号：Q813[生物学—生物工程]