Cationic lipid-mediated transfection of liver cells in primary culture.
We describe transfection of DNA into parenchymal and individual non-parenchymal cell populations from adult rat liver in early primary culture, using cationic lipid as the carrier. All cell populations were transfectable, although lipid requirements varied by cell type and, for hepatocytes, with the age of the culture. For hepatocytes in early primary culture (2-10 hours after plating), pure DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was strikingly more effective than commercial formulations (Lipofectin or TransfectACE) containing components in addition to, or other than DOTMA. For hepatocytes fully adapted to culture (approximately 48 hours after plating), pure DOTMA and Lipofectin were similarly effective. Under optimal conditions, about 10% of hepatocytes expressed the transfected reporter gene. CAT expression in hepatocytes doubled from 48 hours to 7 days after transfection. The effect of culture substratum on transfection efficiency also was examined. The presence of basement membrane-like matrix (EHS gel) reduced uptake of the DNA-lipid complex. However, cells in early culture that were transfected on collagen and then replated on EHS gel, displayed significantly greater reporter gene activity than did cells maintained throughout on collagen. In contrast to hepatocytes, non-parenchymal cells (lipocytes, Kupffer cells and endothelial cells, respectively) were transfected most efficiently by Lipofectin; DOTMA alone was inactive. The methods described will facilitate studies of gene regulation in individual liver cell populations.
Jarnagin
W R
WR
University of California, Liver Center Laboratory, San Francisco 94110.
Debs
R J
RJ
Wang
S S
SS
Bissell
D M
DM
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D008081:Liposomes / Q000494:pharmacology*; D008099:Liver / Q000382:microbiology*; D008297:Male; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection / Q000187:drug effects*
D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium