Construction Of Transgenic Swine Inducibly Expressing Cre Recombinase

In order to knock out genes of human diseases using the Cre/loxP system, we propose to generate Mx1-Cre transgenic swine, in which expression of Cre recombinase is driven by the Mx1 promoter, which is an IFN-inducible promoter expressed mainly in liver and spleen. In order to test the excision activity of the Cre recombinase, the Mx1-Cre transgenic swine line was crossed with a reporter swine line expressing green fluorescent protein (GFP). To generate Mx1-Cre transgenic swine, we constructed a transgenic vector pET28a-Mx1- Cre-BGHpo1yA-FRT2neor. The porcine 468bp Mx1 promoter was amplified from the plasmid pGL3-Mx1, which was a gift from Doctor Annabelle Decreux of Liège university. The porcine 1kb Cre cDNA was synthesized through PCR splicing in our laboratory. The 225bp BGHpolyA fragment was amplified from the vector pcDNA3.1+. The FRT2NeoR cassette was digested with 2 enzymes XholⅠand SalⅠfrom the vector pGC FRT2NeoR, which was a gift from rofessor Stefano Casola of IFOM-Fondazione Istituto FIRC di Oncologia Molecolare. We combined the above 4 fragments through SOE-PCR splicing and restriction enzyme ligation, and then linked the 4 fragments to the prokaryotic expression vector pET28a(+). The recombinant vector was designed with restriction enzymes NheI and NotI, and then the vector was transfected successfully into porcine fibroblast by lipofectamine TM2000. About 10 days after transfection, we selected successfully a monoclone and then checked Cre gene with PCR,integration sits were analyzed by hiTAIL PCR. At last we created transgenic pigs using somatic cell nuclear transfer (SCNT). Transfer of 210, 230, 250, 210, 250, 250 and 215 zygote to 7 surrogates produced 13 piglets. The Cre recombinase expression in transgenic pig is studied by RT-PCR and immunohistochemistry. Mx1-Cre swine harbouring cre gene in genomic DNA is demonstrated by PCR. In conclusion, Mx1-Cre transgenic piglets was successfully produced by SCNT. In conjunction with inducible systems for controlling Cre expression and function, these transgenic swine are likely to have a profound impact on the study of human diseases. Mx1-Cre swine should be of great value for analysis of gene function of human diseases, and to establish animal models of human disorders.