Efficient transformation of mammalian cells using DNA interpolyelectrolyte complexes with carbon chain polycations

Abstract

A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p beta-Gal plasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium+ ++) bromides random copolymer (C16PVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma "Jurkat", and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The in vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of beta-galactosidase (beta-Gal) expression using 4-methylumbeliferril beta-D-galactopyranoside as a substrate (p beta-Gal plasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA affinity to biological membranes is enhanced due to complexation with hydrophobized polycation

“…At the same time, it was shown in the experiments with living cells that complexation of plasmid DNA with both P2 and P2,16 considerably enhanced DNA affinity to living cell membranes [14][15][16]. Such a difference in the behavior of DNA-containing complexes regarding liposomes and cells probably resulted from the rigidity of small liposomes, of which the membranes could be hardly curved without disruption.…”

“…A number of approaches have been developed to introduce DNA into cells [2][3][4][5][6][7][8][9][10][11][12][13]. Recently, for this purpose, it was proposed to incorporate nucleic acids into soluble IPECs with polycations [11][12][13][14][15][16]. Incorporation of DNA into soluble IPEC with quaternized poly-4-vinylpyridine was shown to enhance significantly the efficiency of DNA penetration into Bacillus subtilis cells when compared to native DNA [14][15][16].…”

“…Recently, for this purpose, it was proposed to incorporate nucleic acids into soluble IPECs with polycations [11][12][13][14][15][16]. Incorporation of DNA into soluble IPEC with quaternized poly-4-vinylpyridine was shown to enhance significantly the efficiency of DNA penetration into Bacillus subtilis cells when compared to native DNA [14][15][16]. At the same time, it was shown that amphiphilic oligocations (such as spermine modified by hydrophobic substituents) complexed with DNA effectively stimulated gene transfer into mammalian cells [13].…”

Enhancement of the polycation‐mediated DNA uptake and cell transfection with Pluronic P85 block copolymer

“…This provides for specific and selective binding of the complex with a membrane receptor and DNA uptake into the cells [4,5]. The second approach uses synthetic polycations, for example poly(N-ethyl-4-vinylpyridinium bromide) (PEVP), which permit uptake of the complex and cell transfection in the absence of binding with the receptor [6,7]. Various cell lines have been successfully transfected using either one of these approaches.…”

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Abstract Polyelectrolyte complexes formed between DNA and poly(N-ethyl-4-vinylpyridinium) cations were shown to effectively transfect mammalian ceils [7]. This work suggests that the polycation-mediated uptake of the plasmid DNA and cell transfection are significantly enhanced when these complexes are administered simultaneously with a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymer, Pluronic P85.

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