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. 1992 Aug 25;20(16):4205–4211. doi: 10.1093/nar/20.16.4205

Cationic lipid-mediated transfection of liver cells in primary culture.

W R Jarnagin 1, R J Debs 1, S S Wang 1, D M Bissell 1
PMCID: PMC334127  PMID: 1508714

Abstract

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.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bissell D. M., Arenson D. M., Maher J. J., Roll F. J. Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest. 1987 Mar;79(3):801–812. doi: 10.1172/JCI112887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bissell D. M., Guzelian P. S. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann N Y Acad Sci. 1980;349:85–98. doi: 10.1111/j.1749-6632.1980.tb29518.x. [DOI] [PubMed] [Google Scholar]
  4. Bissell D. M., Hammaker L. E., Meyer U. A. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J Cell Biol. 1973 Dec;59(3):722–734. doi: 10.1083/jcb.59.3.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Brunette E., Stribling R., Debs R. Lipofection does not require the removal of serum. Nucleic Acids Res. 1992 Mar 11;20(5):1151–1151. doi: 10.1093/nar/20.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Camper S. A., Yao Y. A., Rottman F. M. Hormonal regulation of the bovine prolactin promoter in rat pituitary tumor cells. J Biol Chem. 1985 Oct 5;260(22):12246–12251. [PubMed] [Google Scholar]
  8. Caron J. M. Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol. 1990 Mar;10(3):1239–1243. doi: 10.1128/mcb.10.3.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clayton D. F., Darnell J. E., Jr Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol. 1983 Sep;3(9):1552–1561. doi: 10.1128/mcb.3.9.1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Felgner P. L., Ringold G. M. Cationic liposome-mediated transfection. Nature. 1989 Jan 26;337(6205):387–388. doi: 10.1038/337387a0. [DOI] [PubMed] [Google Scholar]
  12. Ferry N., Duplessis O., Houssin D., Danos O., Heard J. M. Retroviral-mediated gene transfer into hepatocytes in vivo. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8377–8381. doi: 10.1073/pnas.88.19.8377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friedman S. L., Roll F. J., Boyles J., Bissell D. M. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8681–8685. doi: 10.1073/pnas.82.24.8681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friedman S. L., Roll F. J. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem. 1987 Feb 15;161(1):207–218. doi: 10.1016/0003-2697(87)90673-7. [DOI] [PubMed] [Google Scholar]
  15. Gabellec M. M., Steffan A. M., Dodeur M., Durand G., Kirn A., Rebel G. Membrane lipids of hepatocytes, Kupffer cells and endothelial cells. Biochem Biophys Res Commun. 1983 Jun 29;113(3):845–853. doi: 10.1016/0006-291x(83)91076-8. [DOI] [PubMed] [Google Scholar]
  16. Ginot F., Decaux J. F., Cognet M., Berbar T., Levrat F., Kahn A., Weber A. Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression. Eur J Biochem. 1989 Mar 15;180(2):289–294. doi: 10.1111/j.1432-1033.1989.tb14646.x. [DOI] [PubMed] [Google Scholar]
  17. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herbst R. S., Friedman N., Darnell J. E., Jr, Babiss L. E. Positive and negative regulatory elements in the mouse albumin enhancer. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1553–1557. doi: 10.1073/pnas.86.5.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Irving M. G., Roll F. J., Huang S., Bissell D. M. Characterization and culture of sinusoidal endothelium from normal rat liver: lipoprotein uptake and collagen phenotype. Gastroenterology. 1984 Dec;87(6):1233–1247. [PubMed] [Google Scholar]
  21. Izban M. G., Papaconstantinou J. Cell-specific expression of mouse albumin promoter. Evidence for cell-specific DNA elements within the proximal promoter region and cis-acting DNA elements upstream of -160. J Biol Chem. 1989 Jun 5;264(16):9171–9179. [PubMed] [Google Scholar]
  22. Koch K. S., Lu X. P., Brenner D. A., Leffert H. L. Differential expression of the transfected liver-specific alpha 1-inhibitor III gene in normal hepatocytes and hepatoma cells in culture. Biochem Biophys Res Commun. 1992 Feb 28;183(1):184–192. doi: 10.1016/0006-291x(92)91626-2. [DOI] [PubMed] [Google Scholar]
  23. Lim K., Chae C. B. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechniques. 1989 Jun;7(6):576–579. [PubMed] [Google Scholar]
  24. Mackett M., Smith G. L., Moss B. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol. 1984 Mar;49(3):857–864. doi: 10.1128/jvi.49.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Muakkassah-Kelly S. F., Jans D. A., Lydon N., Bieri F., Waechter F., Bentley P., Stäubli W. Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor. Exp Cell Res. 1988 Oct;178(2):296–306. doi: 10.1016/0014-4827(88)90400-4. [DOI] [PubMed] [Google Scholar]
  26. Pinkert C. A., Ornitz D. M., Brinster R. L., Palmiter R. D. An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev. 1987 May;1(3):268–276. doi: 10.1101/gad.1.3.268. [DOI] [PubMed] [Google Scholar]
  27. Ponder K. P., Dunbar R. P., Wilson D. R., Darlington G. J., Woo S. L. Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection. Hum Gene Ther. 1991 Spring;2(1):41–52. doi: 10.1089/hum.1991.2.1-41. [DOI] [PubMed] [Google Scholar]
  28. Rippe R. A., Brenner D. A., Leffert H. L. DNA-mediated gene transfer into adult rat hepatocytes in primary culture. Mol Cell Biol. 1990 Feb;10(2):689–695. doi: 10.1128/mcb.10.2.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rosenthal N. Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 1987;152:704–720. doi: 10.1016/0076-6879(87)52075-4. [DOI] [PubMed] [Google Scholar]
  30. Stinski M. F., Roehr T. J. Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components. J Virol. 1985 Aug;55(2):431–441. doi: 10.1128/jvi.55.2.431-441.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thompson J. F., Hayes L. S., Lloyd D. B. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene. 1991 Jul 22;103(2):171–177. doi: 10.1016/0378-1119(91)90270-l. [DOI] [PubMed] [Google Scholar]
  32. Whitlock J. P., Jr, Miller H., Gelboin H. V. Induction of aryl hydrocarbon (benzo(a)pyrene) hydroxylase and tyrosine aminotransferase in hepatoma cells in culture. J Cell Biol. 1974 Oct;63(1):136–145. doi: 10.1083/jcb.63.1.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wilson J. M., Grossman M., Wu C. H., Chowdhury N. R., Wu G. Y., Chowdhury J. R. Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J Biol Chem. 1992 Jan 15;267(2):963–967. [PubMed] [Google Scholar]

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