Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection.
For most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 5-10% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SR alpha, and beta-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin, alpha 1-antitrypsin, or phosphoenolpyruvate carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytes in vivo.
Ponder
K P
KP
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Dunbar
R P
RP
Wilson
D R
DR
Darlington
G J
GJ
Woo
S L
SL
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D005069:Evaluation Studies as Topic; D005786:Gene Expression Regulation; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D015316:Genetic Therapy / Q000379:methods; D008081:Liposomes; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D008081:Liposomes; D011993:Recombinant Fusion Proteins