Transfection of the Albumin Promoter Driven-Oncogenes to Isolated Rodent Hepatocytes, and Transplantation of the Hepatocytes into Livers and Spleens of Syngenic Animals.

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The purpose of this study is to establish a hepatic carcinogenesis model in which the activated oncogenes were transfected into isolated rodent hepatocytes in vitro, and they were subsequently transplanted into the liver or other parts of the body of the syngenic animals. The oncogenes were inserted downstream to the albumin gene promoter, which may facilitate the expression of the transgenes within the hepatocytes. 1)The fragments including core promoters of the albumin gene were amplified by PCR and cloned into a plasmid vector. Using this DNA, we constructed a pAlbElA vector in which ElA was inserted downstream to the albumin promoters. When this vector was introducted into isolated hepatocytes, the gene was transiently expressed in the cells. 2)To efficiently introduce the gene into isolated hepatocytes, a retroviral method was tested using the beta-gal gene as a reporter. We could successfully introduce the beta-gal gene within the cultured newborn rat hepatocytes. 3)Transplantability of isolated hepatocytes was tested using F344 rats and F344 congenic analbuminemic rats. We confirmed the presence of F344 rat hepatocytes within the liver of analbuminemic rats by increase of albumin mRNA and albumin-positive cells within the liver, and elevation of serum albumin 30-90 days after infusion of the cells into the portal vein. 4)Transplantability of the cultured hepatocytes into the livers and spleens was then tested. Primarily-isolated hepatocytes of F344 rats were spheroidally-cultured and transplanted into the livers and spleens of analbuminemic rats. We confirmed that the transplanted hepatocytes maintained normal structures and functions within the organs of the recipients. The beta-gal gene transfected into the isolated F344 rat hepatocytes was temporally expressed within the spleen after transplantation.