猪Oct4-EGFP多能性报告载体的构建与验证

doi:10.16431/j.cnki.1671-7236.2015.08.011

›› 2015, Vol. 42 ›› Issue (8): 1993-1999.doi: 10.16431/j.cnki.1671-7236.2015.08.011

猪Oct4-EGFP多能性报告载体的构建与验证

朱蒙¹, 付博², 刘娣^1,2, 杨秀芹¹

1. 东北农业大学动物科学技术学院, 哈尔滨 150030;
2. 黑龙江省农业科学院畜牧研究所, 哈尔滨 150086

收稿日期:2014-11-03 出版日期:2015-08-20 发布日期:2015-08-27
通讯作者: 杨秀芹 E-mail:xiuqin163@163.com
作者简介:朱蒙(1989-),男,内蒙古赤峰人,硕士生,研究方向:动物遗传育种与繁殖,E-mail:zhupang1118@163.com
基金资助:
国家自然科学基金(31201804、31101700);黑龙江省自然科学基金(QC2013C021);黑龙江省博士后基金(LBr-211032)

Construction and Validation of Oct4-EGFP Pluripotent Reporter Vector in Pig

ZHU Meng¹, FU Bo², LIU Di^1,2, YANG Xiu-qin¹

1. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
2. Institute of Animal Husbandry of Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China

Received:2014-11-03 Online:2015-08-20 Published:2015-08-27

摘要/Abstract

摘要： 本研究旨在构建一套猪Oct4-EGFP多能性报告载体,可在不破坏细胞的前提下研究Oct4的表达规律,从而有利于早期胚胎发育研究及干细胞的研究。试验采用无缝克隆(In-Fusion PCR cloning)技术,将Oct4启动子序列直接重组到pEGFP-N1载体上,用Oct4代替质粒pEGFP-N1中增强型绿色荧光蛋白(EGFP)原有的CMV启动子构建出Oct4-EGFP报告载体,并用脂质体转染技术转染入大白猪胎儿成纤维细胞中,分析Oct4-EGFP报告载体,表达情况。结果发现,经PCR及测序验证,成功构建了Oct4-EGFP多能性报告载体,并在孤雌囊胚上初步验证了载体的有效性;经过脂质体转染,经筛选及PCR鉴定,获得了8株整合有Oct4-EGFP多能性报告载体的转基因细胞。研究结果表明,运用无缝克隆技术可高效率构建Oct4-EGFP多能性报告载体,且获得的转基因阳性细胞可为猪胚胎早期发育和胚胎干细胞研究奠定技术基础。

关键词: 猪; 胚胎; 转基因; 绿色荧光蛋白; 无缝克隆

Abstract: This study aimed to construct Oct4-EGFP pluripotent reporter vector in pig without destroying cells researching the expression pattern of Oct4, thus contributing to early embryonic development and stem cell research.Construct Oct4-EGFP pluripotency reporter vector with the In-Fusion PCR cloning technology, the Oct4 promoter sequence to direct the recombinant vector pEGFP-N1 instead of EGFP original CMV promoter plasmid pEGFP-N1 by Oct4 and liposomal transfection technology, and transfected into livability of porcine fetal fibroblasts cells.The results showed that it had successfully constructed Oct4-EGFP pluripotency reporter vector by PCR and sequencing verified, and initially verified the validity of the carrier in the parthenogenetic blastocysts.After liposomal transfection, 8 genetically modified cells of incorporates pluripotent report carrier with the Oct4-EGFP had been gained by screening and PCR.Thus, the In-Fusion PCR cloning technology could efficiently construct Oct4-EGFP pluripotency reporter vector and obtained transgenic positive cells could lay the foundation for the early development and embryonic stem cell research of pig embryos.

Key words: pig; embryo; genetically modified; enhanced green fluorescent protein; In-Fusion PCR cloning

中图分类号:

Q782

朱蒙, 付博, 刘娣, 杨秀芹. 猪Oct4-EGFP多能性报告载体的构建与验证[J]. , 2015, 42(8): 1993-1999.

ZHU Meng, FU Bo, LIU Di, YANG Xiu-qin. Construction and Validation of Oct4-EGFP Pluripotent Reporter Vector in Pig[J]. , 2015, 42(8): 1993-1999.

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猪Oct4-EGFP多能性报告载体的构建与验证

Construction and Validation of Oct4-EGFP Pluripotent Reporter Vector in Pig

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