EFFICIENT TRANSFER OF REGULATED GENES IN ADIPOCYTES AND HEPATOMA-CELLS BY THE COMBINATION OF LIPOSOMES AND REPLICATION-DEFICIENT ADENOVIRUS

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes , are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times mo re efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepat oma cells with the lipofectAMINE/pSV2-CAT complex in the presence of r eplication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CA T activity of transiently transfected cells was increased about 50-fol d when compared to the calcium phosphate procedure. To determine wheth er this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a const ruct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykina se gene fused to the CAT gene (pPL1-CAT). This construct was shown pre viously to be cAMP-responsive after calcium-phosphate-mediated transfe ction of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in whic h pPL1-CAT was either transiently or stably transferred by lipofectAMI NE and adenovirus responded to isoproterenol or cAMP, respectively, wi th a 2-3-fold increase in CAT activity. Therefore the association of l iposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.