James R. Jacob

Immortalization of chimpanzee hepatocytes with an amphoteric retrovirus encoding simian virus 40 T antigen

Primary chimpanzee (Pan troglodytes) hepatocyte cultures were maintained in a serum-free medium containing hormones and growth factors and exhibited the de novo synthesis and secretion of numerous liver-specific plasma proteins for over 3 weeks in vitro. The long-term maintenance of differentiated, primate hepatocytes in this serum-free medium allowed for subsequent immortalization events to occur after infection with the amphoteric retrovirus U19, which encodes the simian virus 40 large T antigen oncogene. Several hepatocyte cell lines were selected and examined for the expression of liver-specific plasma proteins and the capacity to synthesize apolipoproteins. Several cell lines expressed a majority of the plasma proteins investigated, including apolipoproteins A1 and E. These results demonstrate the ability of this serum-free medium to maintain long-term differentiated primate hepatocytes, allowing for the experimental immortalization of this cell type in vitro and the maintenance of differentiated functions in the established cell lines. This methodology should be amenable to the study of the liver and its related diseases.

Genomic organization of the simian virus 40-adenovirus 7 hybrid virus, PARA(cT), that encodes a nuclear transport defective simian virus 40 T antigen

The genomic organization of the simian virus 40 (SV40)-adenovirus (Ad)7 hybrid virus, PARA(cT), was examined. A deletion of approximately 5529 bp of Ad7 DNA extends from 78.8 map units to 94.0 map units and is replaced by an SV40 DNA insert of 3809 bp. The left-hand end of the insertion begins at SV40 nucleotide 5168, 5 bp upstream of the ATG initiation codon for T-ag synthesis. The sequence extends counterclockwise through the T-ag encoding sequences and into SV40 late region DNA. Most of the late region DNA has been removed in a deletion between nucleotides 2464 and 301. One of the 72-bp repeats has also been deleted. The right-hand end of the SV40 DNA insert is at nucleotide 4366. Thus, a portion of the SV40 DNA early region is repeated at both ends of the insert (nucleotides 5168-4366).