A simple and rapid nonviral approach to efficiently transfect primary tissue–derived cells using polyethylenimine

Abstract

This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.

Linear polyethylenimine produced by partial acid hydrolysis of poly(2-ethyl-2-oxazoline) for DNA and siRNA delivery in vitro

“…The ability of PEI to deliver DNA and siRNA both in vitro and in vivo was reported recently. [22][23][24] Our transfection experiment with PEI70-DNA demonstrated that similar transfection efficiency as with Lipofectamine 2000 can be obtained while lowering cytotoxicity. The influence of N/P ratio of PEI to DNA and varying hydrolysis percentages of PEtOx was explored.…”

An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

“…In a reported case, the authors showed an optimized protocol for more efficient transfection of adherent primary mammalian cells using 25 kDa branched PEI. 12 In this study, the modified method was applicable for, at least, PEI and liposome to deliver genes into a wider range of cell lines.…”

“…In general, complexes may undergo physicochemical changes when mixed at different concentrations, and the aggregation of particles may also be influenced by the different ionic strength in the medium. 12,35 The complexation of pDNA/material particle was formed in 700 μL solution for the modified transfection while in 500 μL and then diluted to 2,500 μL system for standard transfection, leading to a different particle concentration and size. Altogether, the change of cellular endocytosis pathway, decrease of particle size, and increase of particle concentration made the modified transfection method a potent tool for nonviral material-mediated gene delivery into a wide range of cell strains.…”

“…11 The gene delivery efficiency of nonviral materials is usually influenced by many factors, such as the quantity and quality of deoxyribonucleic acid (DNA), ratio of DNA to material, serum, antibiotics, solvent for DNA and material, incubation time, and mixing order. [12][13][14][15][16][17][18] Therefore, the readily available off-the-shelf gene carriers usually provide standard transfection procedures that have been considered to generate the highest gene transfection efficiency for cultured cells. 19,20 Branched polyethyleneimine (PEI, Mw~=25 kDa), a typical cationic polymer, has been commonly used as the 'gold standard' for in vitro gene delivery.…”

Transfecting CHO-K1 Cells: Comparison of CaPO4, Electroporation and Lipoplex Method with In-house Prepared Polyplex

“…In comparison to viral vectors, non-viral transfection methods such as electroporation, calcium phosphate (CaPO 4 ) co-precipitation, lipoplex and polyplexmediated methods have been benefited in gaining regulatory clearance and thus preferred for therapeutic protein production as well as for gene therapy [6] . However, these non-viral methods vary with respect to transfection efficiency (Table 1) [7][8][9][10][11] and production cost. Thus, it is important to select an appropriate transfection method, which will be highly efficient and economic.…”