COMPLEMENTATION OF THE GENETIC-DEFECT IN GUNN RAT HEPATOCYTES IN-VITRO BY HIGHLY EFFICIENT GENE-TRANSFER WITH CATIONIC LIPOSOMES

In this article, we report complementation of the genetic defect of is olated Gunn rat hepatocytes by a highly efficient method for lipofecti on. Transfections were performed 24 h after plating by using the catio nic liposome DOTAP. On average, transfection efficiencies of 21% lacZ cells with WAg/Rij rat cells and 27% lacZ(+) cells with Gunn rat cells could be obtained when the parenchymal cells were transfected in a ho rmone-defined, serum-free medium. LacZ expression vectors with the CMV promoter were more effective than constructs containing the RSV or th e TK promoter. A linear relationship between the viability of hepatocy tes after isolation and the percentage of lacZ cells was observed with both rat strains, with a maximum of 40% lacZ(+) cells at a viability of 94%. The transfection efficiencies were significantly lower in the absence of growth factors, in dexamethasone-containing medium, or when serum was present during plating. Our data are consistent with the as sumption that a mitotic event is required for efficient lipofection. B ilirubin conjugation activity could be detected in microsomes from Gun n rat hepatocytes after transfection with two different B-UDPST expres sion contructs. Highest conjugation activity was achieved with a vecto r containing a terminal intron. With this construct on average 4% of t he bilirubin conjugation activity of normal human liver microsomes cou ld be achieved in total microsomes of transfected Gunn rat hepatocytes . The implications of our data for gene therapy of hepatic disease wit h nonviral vectors, in particular bilirubin conjugation deficiency (Cr igler-Najjar disease) are discussed.