| Cre (causes recombination) is the reorganization of the Cre gene encoding a 38 KD protein, derived from P1 phage in 1029 bp section of the protein coding sequences, which can identify specific LoxP target point and on its catalytic fracture and recombination, resulting in the precision of a class of recombinant DNA enzyme, which can play a significant role in gene regulation and deletion. Cre/LoxP has been applicated widely in mammals. Because the swine and the human are extremely similar in size, physiology, tissue development, diseases progress, genome and chorosome structure, the transgenetic pig can be applied as a modle in human diseases research.The final aim of the study is to use transgenic technology and animal cloning technology to build the human pig liver disease model, so we choose the fibroblast of small swine as recipient cell. We constructed the transgenetic pig fibroblast expressed the specific Cre recombinase in liver byα1-anti-trypsin promoter (Human alpha-1-antitrypsin promoter, pHAAT).This study amplified the liver specific promoter- pHAAT gene form blood, and then through integration of triple PCR technology, we put pHAAT gene, Cre recombinant gene and BGHpolyA integration and the successful full-length 3906 bp of pHAAT-Cre - BGHpolyA integration fragment was abtained. This was followed by the successful construction of a reorganization of Cre expression vector pET28a-pHAAT-Cre-BGHpolyA-FRT2neor. We transfected the linear vector into the procine froblast through LipofectamineTM2000 mediated and selected with 250μg/ml G418 for 7 to 10 days. Using the specific primers of pHAAT gene and the Cre gene for PCR identification with the positive cells. The electrophoresis exhibited that there were 1272bp and 1044bp fragments. Which demonstrated the pHAAT gene and the Cre gene inserted in cells genome. Access to the success of the recombination of the liver-specific expression of Cre transgenic pig fibroblast.
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