Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer

Abstract

A systematic investigation of factors influencing the efficiency of polybrene-assisted gene transfer for both transient and stable foreign gene expression was carried out utilizing NIH 3T3 fibroblasts as prototypic recipients for the plasmid expression vectors pSV2cat and pSV2neo. While transfection cocktail composition and cell density, in addition to polybrene exposure conditions and exogenous DNA concentration, each played an important role, the key determinant to achieving excellent transfection efficiency proved to be the DMSO treatment regimen. Under optimal conditions, the yield of colonies resistant to the neomycin analog, G418, increased linearly at the rate of 10 clones/ng of input (native form I pSV2neo) DNA up to a plasmid concentration of 50 ng, whereupon the dose-response for colony recovery became semilogarithmic. The incidence of stable transformants was doubled by linearization of the vector DNA, whereas the addition of carrier DNA to the transfection cocktail was without effect until present at concentrations above 10-fold molar excess, at which point the efficacy of gene transfer declined rapidly. Combined Southern and dot-blot analyses of transformed cell DNA demonstrated that the polybrene-DMSO procedure led to the stable integration of relatively few copies of the marker gene in each transformant; the actual number varied from 1–3 to 10–15 per host genome, depending on the concentration of pSV2neo DNA added. The potential for the adaptation of this DNA transfection procedure for general use with other mammalian cell types, as well as its technical strengths and weaknesses, is discussed.