Cationic lipid-mediated transfection of liver cels in primary culture

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.

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…Many different gene delivery methods are available including cationic lipid-based DNA transfection [44][45][46][47][48][49] and retroviral and adenoviral gene transfer vectors; [50][51][52][53][54][55] however, the use of these methods for hepatocytes has not been effective because hepatocytes do not divide, are highly susceptible to toxicity, and transfection efficiencies in hepatocytes are extremely low (HC Isom, unpublished data). 11 Previous studies have reported baculovirus-mediated gene delivery to primary hepatocytes.…”

Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER

“…[5][6][7] The process of lipoplex formation involves electrostatic interactions between the cationic lipid and anionic DNA, as opposed to traditional liposome technologies which result in encapsulation of DNA. 8 Since their introduction 10 years ago, 9 many cationic lipid formulations have been tested in a variety of in vitro situations and have often yielded highly efficient transfection, 10,11 superior to that obtained with encapsulating liposomes. 12 However, studies administering lipoplexes to a range of tissues in vivo have led to mixed results.…”

Transient Disruption of Intercellular Junctions Enables Baculovirus Entry into Nondividing Hepatocytes

“…Although a large number of cationic lipid-based DNA transfection methods are available (6,16,17,24,30,39), those that we have tested on primary rat hepatocyte cultures result in very low transgene expression (approximately 1 to 3% [H. C. Isom, unpublished data]). The use of viral gene transfer vectors in primary hepatocyte cultures is promising, but each method has drawbacks.…”