骨骼肌特异性表达猪FSⅠ-Ⅰ转基因猪胚胎成纤维细胞克隆的构建与鉴定

J4 ›› 2012, Vol. 38 ›› Issue (2): 192-196.

骨骼肌特异性表达猪FSⅠ-Ⅰ转基因猪胚胎成纤维细胞克隆的构建与鉴定

袁婷|刘帅|李小平|唐成程|韩晓蕾|逄大欣|欧阳红生

（吉林大学畜牧兽医学院动物胚胎工程吉林省重点实验室|吉林长春130062）

收稿日期:2011-09-28 出版日期:2012-03-28 发布日期:2012-03-28
通讯作者: 欧阳红生 E-mail:（Tel：0431-87963175,E-mail：biotechs64@163.com）
作者简介:袁婷(1986-)|女|四川省长宁县人|在读理学硕士|主要从事分子生物学和细胞生物学的研究。
基金资助:
国家自然科学基金资助课题(30871841)

Construction and identification of skeletal muscle-specific FS Ⅰ-Ⅰ expression transgenic porcine embryonic fibroblast lines

YUAN Ting,LIU Shuai,LI Xiao-ping,TANG Cheng-cheng,HAN Xiao-lei|PANG Da-xin,OUYANG Hong-sheng

(Jilin Provincial Key Laboratory of Animal Embryo Engineering,College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

Received:2011-09-28 Online:2012-03-28 Published:2012-03-28

摘要/Abstract

摘要：

目的：构建骨骼肌特异性表达猪FSⅠ-Ⅰ真核表达载体，转染猪胚胎成纤维细胞并筛选整合有FSⅠ-Ⅰ基因的细胞克隆，为研究FSⅠ-Ⅰ对猪骨骼肌的影响奠定基础。方法：通过RT-PCR方法从猪骨骼肌中扩增FS A（包含FS N端和结构域FSⅠ）和结构域FSⅠ；利用PCR方法分别从鼠基因组、人血液基因组和pcDNA3.1（+）载体上扩增出MCK增强子、ACTA 1启动子和BGHpolyA。将上述4个片段分别连接到真核表达载体pGKneotpAlox2上，构建骨骼肌特异性表达FSⅠ-Ⅰ（包含FS N端和2个连续FSⅠ结构域）的载体pGK-MCK-ACTA 1-FSⅠ-Ⅰ-BGHpolyA；用FuGENE
R　HD转染猪胚胎成纤维细胞，经药物G418筛选和PCR鉴定阳性克隆。结果：成功构建骨骼肌特异性表达猪FSⅠ-Ⅰ的表达载体pGK-MCK-ACTA1 promoter-FSⅠ-Ⅰ-BGHpolyA，并成功整合到猪胚胎成纤维细胞的基因组中，获得了整合有目的基因FSⅠ-Ⅰ的猪胚胎成纤维细胞克隆。结论：获得了骨骼肌特异性表达猪FSⅠ-Ⅰ的猪胚胎成纤维细胞克隆，为通过核移植方法获得表达FSⅠ-Ⅰ转基因猪提供了供体细胞。

关键词: 卵泡抑素；猪胚胎成纤维细胞；真核表达

Abstract:

To construct the skeletal muscle-specific FSⅠ-Ⅰ eukaryotic expression vector and identify the integration of FSⅠ-Ⅰ gene in porcine embryonic fibroblasts,and to provide a basis for study on the effect of FSⅠ-Ⅰ on skeletal muscle.Methods The FS A (containing FS N and FSⅠ) and FSⅠ were amplified by RT-PCR from porcine skeletal muscle;the MCK enhancer,ACTA1 promoter and BGHpolyA were amplified by PCR from mouse genenome,human genenome and pcDNA3.1(+), respectively.The four fragments mentioned above were subcloned into the eukaryotic expression vector pGKneotpAlox2,and then the vector was transfeced into porcine embryonic fibroblasts using the FuGENER　HD reagent,according to the manufacturer’s protocol.The cells were selected with G418 antibiotic and identified by PCR amplification.Results The FS Ⅰ-Ⅰ skeletal muscle -specific expression vector was successfully constructed and integrated to the genome of the porcine fibroblasts and the clone cells integrating the FSⅠ-Ⅰ gene were obtained.Conclusion The porcine fibroblast clone expressing FSⅠ-Ⅰ is obtained and it should be of great value to the construction of FSⅠ-Ⅰ transgenic swine.

Key words: follistatin；porcine embryonic fibroblasts；eukaryotic expression

中图分类号:

S852

袁婷|刘帅|李小平|唐成程|韩晓蕾|逄大欣|欧阳红生. 骨骼肌特异性表达猪FSⅠ-Ⅰ转基因猪胚胎成纤维细胞克隆的构建与鉴定[J]. J4, 2012, 38(2): 192-196.

YUAN Ting,LIU Shuai,LI Xiao-ping,TANG Cheng-cheng,HAN Xiao-lei|PANG Da-xin,OUYAN. Construction and identification of skeletal muscle-specific FS Ⅰ-Ⅰ expression transgenic porcine embryonic fibroblast lines[J]. J4, 2012, 38(2): 192-196.