title: Optimization of gene transfer using cationic lipids in cell lines and primary human CD4+ and CD34+ hematopoietic cells.
abstract: 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.
authors.affiliation: University of Colorado Health Sciences Center Division of Medical Oncology, Denver 80262, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Harrison
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Tomczak
authors.forename: C
authors.initials: C
authors.lastname: Hogan
authors.forename: E J
authors.initials: EJ
authors.lastname: Shpall
authors.forename: T J
authors.initials: TJ
authors.lastname: Curiel
authors.forename: P L
authors.initials: PL
authors.lastname: Felgner
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D002412:Cations; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids; D008156:Luciferases / Q000235:genetics; D008223:Lymphoma; D051379:Mice; D010714:Phosphatidylethanolamines / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: C098223:2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate; D018952:Antigens, CD34; D002412:Cations; D008055:Lipids; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; D013096:Spermine; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D008156:Luciferases