Production of transgenic pigs for the use of bioartificial liver.

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Producing transgenic cattle that can secret human proteins is a promising strategy for development of a bioreactor for human pharmaceuticals or bioartificial liver support. We constructed pCX-hAlb-EGFP, that is, a combined gene of human albumin cDNA and EGFP cDNA connected with CMV-IE enhancer, chicken β-actin promoter, and rabbit β-globin poly A signal for the purpose of producing a transgenic pig that can secret human albumin systemically. NIH3T3 cells transformed with pCX-hAlb-EGFP by the lipofection method showed intense expression of human albumin and EGFP genes. Thus, we introduced it into porcine oocytes using sperm vector method, and a piglet was produced which showed clear expression of GFP in the hooves and skin. PCR analysis of genomic DNA extracted from several tissues including the liver showed that they possessed the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the same tissues. The transgene was integrated and expressed in the porcine liver, which is indispensable for our purpose of utilizing the transgenic liver for bioartificial liver support. Real time RT-PCR analysis exhibited that the expression rate of human albumin gene and EGFP gene against β-actin promoter in the liver showed adequate quantity of transcribed mRNA and fairly good expression rates as compared with those of the other tissues. These results will promise successful result of further trials of producing desirable transgenic pig with the liver secreting human albumin.