Efficient DNA-mediated gene transfer into primary cultures of adult rat hepatocytes

DNA
D S Pasco, J B Fagan

Abstract

An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes. Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells. Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured. Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein. Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability. (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential. (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel. (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities. (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times. (v) Length of time hepatocytes were maintained in culture before initiating transfect...Continue Reading

References

Nov 24, 1979·Nucleic Acids Research·H C Birnboim, J Doly
Jun 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·J M WilsonR C Mulligan
May 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·J M WilsonR C Mulligan
Apr 1, 1986·In Vitro Cellular & Developmental Biology : Journal of the Tissue Culture Association·B L KreamerH C Pitot
May 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·J A WolffH Leffert
Nov 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·K SogawaY Fujii-Kuriyama
Feb 1, 1986·Molecular and Cellular Biology·R Tur-KaspaD A Shafritz
Aug 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·F D LedleyS L Woo
Aug 1, 1987·Molecular and Cellular Biology·C Chen, H Okayama
Jan 1, 1987·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·M FujitaL M Reid
May 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·H C IsomJ Mummaw
Apr 1, 1973·Virology·F L Graham, A J van der Eb
Nov 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·L A LaiminsP Gruss
Nov 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·C M GormanB H Howard
Mar 11, 1983·Nucleic Acids Research·H Luthman, G Magnusson
Sep 1, 1984·Molecular and Cellular Biology·D M JeffersonL M Reid
Sep 1, 1982·Molecular and Cellular Biology·C M GormanB H Howard
Jan 1, 1980·Annals of the New York Academy of Sciences·D M Bissell, P S Guzelian
Sep 1, 1981·Somatic Cell Genetics·C M Corsaro, M L Pearson

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Citations

May 1, 1992·In Vitro Cellular & Developmental Biology : Journal of the Tissue Culture Association·A P LiD L Kaminski
Jun 1, 1996·The International Journal of Biochemistry & Cell Biology·C M HilliardG C Yeoh
Feb 1, 1995·The Journal of Clinical Investigation·J K WestwickD A Brenner
Mar 1, 1992·In Vitro Cellular & Developmental Biology : Journal of the Tissue Culture Association·N A BetzJ C Pelling
May 1, 1995·Molecular Carcinogenesis·J A Silverman, B A Hill
Aug 18, 2010·Advanced Drug Delivery Reviews·Barbara Bonamassa, Dexi Liu
Mar 1, 1992·The Journal of Surgical Research·J RozgaA A Demetriou
Feb 1, 1993·Current Opinion in Genetics & Development·M Grossman, J M Wilson
May 16, 2012·Critical Reviews in Toxicology·Edward L LeCluyseMark J Powers
Apr 19, 1996·The Journal of Biological Chemistry·P HonkakoskiM Negishi
Feb 1, 1992·Journal of Virology·H E BlumJ R Wands

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