Abstract
In this article, we report complementation of the genetic defect of isolated Gunn rat hepatocytes by a highly efficient method for lipofection. Transfections were performed 24 h after plating by using the cationic liposome DOTAP. On average, transfection efficiencies of 21% lacZ+ cells with Wag/Rij rat cells and 27% lacZ+ cells with Gunn rat cells could be obtained when the parenchymal cells were transfected in a hormone-defined, serum-free medium. LacZ expression vectors with the CMV promoter were more effective than constructs containing the RSV or the TK promoter. A linear relationship between the viability of hepatocytes after isolation and the percentage of lacZ+ cells was observed with both rat strains, with a maximum of 40% lacZ+ cells at a viability of 94%. The transfection efficiencies were significantly lower in the absence of growth factors, in dexamethasone-containing medium, or when serum was present during plating. Our data are consistent with the assumption that a mitotic event is required for efficient lipofection. Bilirubin conjugation activity could be detected in microsomes from Gunn rat hepatocytes after transfection with two different B-UDPGT expression constructs. Highest conjugation activity was achieved with a vector containing a terminal intron. With this construct on average 4% of the bilirubin conjugation activity of normal human liver microsomes could be achieved in total microsomes of transfected Gunn rat hepatocytes. The implications of our data for gene therapy of hepatic disease with nonviral vectors, in particular bilirubin conjugation deficiency (Crigler–Najjar disease) are discussed.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Author information
Authors and Affiliations
Additional information
Department of Gastroenterology, Sophia Children’s Hospital Rotterdam, The Netherlands
Rights and permissions
About this article
Cite this article
Wilke, M., Bijma, A., Timmers-Reker, A. et al. Complementation of the genetic defect in Gunn rat hepatocytes in vitro by highly efficient gene transfer with cationic liposomes. Gene Ther 4, 1305–1312 (1997). https://doi.org/10.1038/sj.gt.3300542
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3300542