1-2-dielaidoylphosphatidylethanolamine and Lymphoma

Cationic lipids offer several advantages for gene delivery, both in vitro and in vivo. However, high-efficiency gene transfer has been demonstrated only for limited cell types. Here, we examine the level of expression of a luciferase reporter gene, delivered using cationic lipids, in both cell lines and primary human cells including peripheral blood mononuclear cells and CD34(+)-enriched hematopoietic cells. Variables shown to affect the efficiency of gene expression included the type of lipid, the amounts of DNA and lipid, the day of assay following transfection, the media used for lipid:DNA complex formation, the cell number, the promoter driving expression of the reporter gene and the physiological state of the cells (e.g., whether or not cells were differentiated). The maximal luciferase expression observed with the primary cells was one to two orders of magnitude lower than that seen in cell lines. Further studies, possibly involving altering the growth conditions for the cells, or using episomal vectors that will allow extrachromosomal maintenance of the DNA, are required to improve the level of transgene expression in the primary human cell types used here.

Topics: Animals; Antigens, CD34; Cations; CD4-Positive T-Lymphocytes; Cell Line; DNA; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Genetic Techniques; Genetic Vectors; Hematopoietic Stem Cells; Humans; Lipid Metabolism; Lipids; Luciferases; Lymphoma; Mice; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Spermine; Tumor Cells, Cultured

In this chapter, we describe the use of recombinant VV to express G alpha subunits efficiently in S49 cyc- cells, a murine lymphoma cell line deficient in endogenous Gs alpha activity. Additional studies have shown that VV infection does not interfere with G-protein-coupled signal transduction events. In particular, the VV-expressed rat Gs alpha subunit is able to mediate efficient receptor-dependent and receptor-independent activation of adenylyl cyclase in cyc- cells. This system should therefore be useful for rapidly assessing the activity of modified or chimeric Gs alpha subunits.

Topics: Animals; Cell Line; Chlorocebus aethiops; Drosophila; Gene Expression; Genetic Vectors; GTP-Binding Proteins; HeLa Cells; Humans; Lymphoma; Mammals; Mice; Phosphatidylethanolamines; Recombinant Proteins; Transfection; Vaccinia virus