Characterization of the interplay between the main factors contributing to lipoplex-mediated transfection in cell cultures.
Transfection efficiency of lipoplex-mediated gene delivery is multifactorial. However, the mode of interaction between the factors which affect transfection is not fully understood. To help fill this deficiency we evaluated the effect of the interplay between several variables that affect transfection efficiency in cell cultures. For this, we applied the Analysis of Variance Model with Fixed Effects and Repeated Measures to assess the data. The variables studied include: two different genes, Luc, and human growth hormone (hGH), in three different plasmids (two of which contain the luciferase (Luc) gene, but different promoter-enhancer regions (CMV and H19) and one plasmid coding hGH with a S16 promoter); three topoisoforms of pDNA (supercoiled (SC), open circular (OC), and closed circular (CC)); three cationic lipid compositions, all based on the monocationic lipid DOTAP (100% DOTAP, DOTAP/DOPE 1 : 1, and DOTAP/cholesterol 1 : 1, all ratios are mole ratios); two DNA-/L+ charge ratios (0.2 and 0.5); and two cell lines (NIH 3T3 and MBT-2). Our statistical analysis confirmed that the cell type, the gene used for transfection, the promoter type, the type of helper lipid, and DNA-/DOTAP+ charge ratio, all affect transfection efficiency in a statistically significant manner. The most efficient lipoplex formulation in both cell lines was that based on DOTAP (without helper lipid), having CC plasmid DNA. We suggest that for obtaining the most transfection-efficient lipoplex one should select the best topoisoform of pDNA for each particular cell type, and complex it with cationic liposomes having optimal lipid composition.
Boktov
Julia
J
Laboratory of Membrane and Liposome Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Hirsch-Lerner
Danielle
D
Barenholz
Yechezkel
Y
D016475:3T3 Cells; D000818:Animals; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D019382:Human Growth Hormone; D004247:DNA; D008156:Luciferases