PiggyBac转座子介导的转FGF5s基因绒山羊胎儿成纤维细胞的制备  

Cloning of FGF5s Gene from Cashmere Goat and Its Stable Transfection of Caprine Fetal Fibroblasts Based on PiggyBac Transposon

作　　者：何晓琳[1] 袁超[1] 屈雷[2] 陈玉林[1] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]榆林学院生命科学研究中心,榆林719000

出　　处：《畜牧兽医学报》2013年第12期1926-1931,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重大转基因专项(2011ZX08008-002);国家自然科学基金(31101684);西北农林科技大学拔尖人才支持计划

摘　　要：本研究旨在建立可稳定表达FGF5s的绒山羊胎儿成纤维细胞系。采用RT-PCR方法,从陕北白绒山羊皮肤组织cDNA中克隆FGF5s基因,构建PB转座子介导的真核表达载体PB-EGFP-FGF5s;在脂质体的介导下,将PB-EGFP-FGF5s载体和转座酶载体共转染绒山羊胎儿成纤维细胞,通过嘌呤霉素进行稳定筛选。并利用PCR和RT-PCR鉴定FGF5s基因在细胞基因组中的整合及表达情况。结果表明,成功克隆了陕北白绒山羊FGF5s基因并构建了PB-EGFP-FGF5s真核表达载体;PCR结果显示,FGF5s已整合到细胞基因组中;RT-PCR结果表明,该基因在转录水平上表达。另外,转染试验表明PB转座子可在绒山羊胎儿成纤维细胞中发生高效转座。本研究为制备绒山羊转基因细胞系提供了一种高效的方法,并为下一步通过核移植方法获得转基因绒山羊奠定了基础。Abstract. The objective of the present study is to establish the Cashmere goat fetal fibroblasts cell line that stably expresses Cashmere goat FGF5s. By using RT-PCR technology, the FGFSs gene of Cashmere goat was first cloned from total RNAs which extracted from Cashmere goat skin, and then inserted into the eukaryotic expression vector PB-CMV-Puro-EGFP based on PiggyBac transposon. PB-EGFP-FGF5s plasmid and PB transposase plasmid were used to co-transfect the Cashmere goat fetal fibroblasts by LipofectamineTM2000. A stable fibroblast line which expressed green fluorescence was obtained using Puromycin screening. The integration of exogenous DNA and the exogenous mRNA expression of FGFSs were identified through PCR and RT-PCR, re spectively. The complete CDS area of FGFSs was cloned and then the PB-EGFP-FGF5s plasmid was constructed successfully. The present results of the transgenic cell clones identification illus trated that the exogenous FGFSs gene was integrated into the Cashmere goat genome. Moreover, mRNA of FGFSs was detected to express in fibroblast cell line according to the RT-PCR data. Collectively, this study provides an effective approach for preparing transgenic cells of Cashmere goat, and a basis for nuclear transplantation in the future.

关 键 词：陕北白绒山羊 FGF5s PIGGYBAC转座子 稳定转染 

分 类 号：S827[农业科学—畜牧学] S814.8[农业科学—畜牧兽医]