Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes.
In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
Hilliard
C M
CM
Department of Biochemistry, University of Western Australia, Nedlands, Australia.
Fletcher
S
S
Yeoh
G C
GC
D000818:Animals; D002130:Calcium Phosphates; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003637:DEAE-Dextran; D004247:DNA / Q000032:analysis; D018274:Electroporation; D005314:Embryonic and Fetal Development / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D008081:Liposomes; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D010714:Phosphatidylethanolamines; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection
D002130:Calcium Phosphates; D008081:Liposomes; D010714:Phosphatidylethanolamines; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C100004:transfectACE; C485838:calcium phosphate, monobasic, anhydrous; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D003637:DEAE-Dextran; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous