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fat-1基因脂肪组织特异性表达载体的构建 及其山羊转基因细胞系的建立
陈建文,刘星,桂涛,李运生,章孝荣,张瑾,张运海
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(安徽地方畜禽遗传资源保护与生物育种省级实验室,安徽农业大学动物科技学院,合肥230036;河北科技师范学院生命科学学院,秦皇岛066000)
摘要:
旨在构建一种筛选标记可全部去除的脂肪组织特异性表达fat-1基因的载体,将其转染山羊胎儿成纤维细胞,筛选出稳定整合fat-1基因的转基因细胞系。首先将人工合成的fat-1基因连接至L28-Wnt10b载体(1种带有小鼠脂肪组织特异性启动子Fabp4的载体)上,构建成fat-1基因脂肪组织特异性表达载体L28-fat1;同时经多次克隆构建成1种筛选标记可全部去除的骨架载体MCS-3s-LoxP-RFP;然后,利用Hind III和Not I对上述2种载体进行双酶切,接着进行连接,构建出筛选标记可全部去除的脂肪组织特异性表达fat-1基因的表达载体。采用脂质体介导的方法转染山羊胎儿成纤维细胞,通过G418筛选转基因细胞。酶切鉴定及PCR检测结果表明,成功构建了3s-LoxP-RFP-FABP4-fat1表达载体,并首次获得了脂肪组织特异性表达fat-1基因的山羊胎儿成纤维转基因细胞系,为将来通过体细胞核移植创制脂肪组织特异表达fat-1基因的优质肉用转基因山羊新材料奠定了基础。
关键词:  不饱和脂肪酸脱氢酶  脂肪组织特异性表达  脂肪酸结合蛋白4启动子  转基因山羊  标记去除
DOI:
基金项目:转基因生物新品种培育科技重大专项(2009ZX08006-007B)资助。
Construction of fat-1 adipose tissue specific expression vector and production of goat transgenic fibroblast cell line
CHEN Jian-wen,LIU Xing,GUI Tao,LI Yun-sheng,ZHANG Xiao-rong,ZHANG Jin,ZHANG Yun-hai
(Anhui Provincial Lab for Local Livestock and Poultry Genetic Resource Conservation and Biobreeding, School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036;School of Life Science and Technology, Hebei Normal University of Science & Technology, Qinhuangdao 066000)
Abstract:
The aim of this study is to construct a marker removable, fat-1 adipose tissue specific expression vector and produce the transgenic goat fibroblast cell line for nuclear transfer. Firstly, the fat-1 gene was synthezised and a fat-1 adipose tissue specific expression vector was constructed. Secondly, the adipose tissue specific expression cassette was subcloned into a marker removable backbone vector (MCS-3s-LoxP-RFP) to construct a fat-1 marker removable adipose tissue specific expression vector driven by mouse Fabp4 promoter. Finally, the goat fetal fibroblasts was transfected with the vector by LipofectmineTM?2000?and selected in medium with G418?for two weeks, and then G418 resistant transfectants were identified by PCR. The results showed that the fat-1 marker removable adipose tissue specific expression vector was successfully constructed and the transgenic goat fibroblast cell lines were well established. It would pave the way for obtaining the marker-free fat-1 transgenic goat by SCNT.
Key words:  fat-1  adipose tissue specific expression  Fabp4 promoter  transgenic goat  marker-free

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