肝特异性表达Cre重组酶转基因猪成纤维细胞的建立

试验旨在建立肝脏特异性表达Cre重组酶猪成纤维细胞系,为获得肝脏特异性表达Cre重组酶转基因猪奠定基础。通过PCR方法分别从血液和pcDNA3.1(+)载体上扩增肝脏特异性启动子α1-抗胰蛋白酶启动子(PhAAT)和BGHpolyA片段。通过双酶切(XhoⅠ、SalⅠ)从载体pGC-FRT2neo上获得FRT2neo交换盒,再利用重叠PCR方法获得猪源的Cre重组酶基因,最后通过三重融合PCR方法和酶切方法将4个片段连接。利用真核表达载体pC1-neo构建出Cre表达载体pC1-PhAATCreBGHpolyA-FRT2neo。Lipofectamine^TM2000介导转染猪成纤维细胞,通过G418筛选出阳性细胞系,最后通过PCR鉴定证明构建的Cre重组酶表达载体pC1-PhAATCreBGHpolyA-FRT2neo成功整合到细胞的基因组中,获得了肝脏特异性表达Cre重组酶猪成纤维细胞,为最终通过核移植方法获得肝脏特异性表达Cre重组酶转基因猪奠定基础。

Establishment of Liver Specific Expression of Cre Recombinase Transgenic Pig Fibroblast

To obtain the transgenic swine of liver specific expression Cre recombinase, we constructed porcine fibroblast line of liver specific expression Cre recombinase. The promoter of alpha1-human antitrypsin and the BGHpolyA were amplified respectively from the homo-blood genome and the vector of pcDNA3.1(+) through PCR . Cre recombinase gene of porcine origian was obtained by the method of overlap PCR. The FRT2neo cassette was digested with XhoⅠ and SalⅠ from the vector pGC-FRT2neo, which was awarded by professor Stefano Casola of Italy. We combined the above four fragments through SOE-PCR splicing and restriction enzyme ligation, and then linked the four fragmens to the eukaryotic expression vertor pC1-neo. The recombinant was transfected into porcine fibroblast by Lipofectamine^TM2000. Successfully constructed the Cre recombinase expression vector and integrated to the genome of the porcine fibroblast and obtained the fibroblast of liver specific expression Cre recombinase. The obtaining of the porcine fibroblast of liver specific expression Cre recombinase should be of great value to the construction of the liver specific expression Cre recombinase transgenic swine.