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Comparative Study
. 2009 Jul 23;14(7):2669-83.
doi: 10.3390/molecules14072669.

Mechanism of introduction of exogenous genes into cultured cells using DEAE-dextran-MMA graft copolymer as non-viral gene carrier

Yuki Eshita  1 , Junko Higashihara, Masayasu Onishi, Masaaki Mizuno, Jun Yoshida, Tomohiko Takasaki, Naoji Kubota, Yasuhiko Onishi
Affiliations
Comparative Study

Mechanism of introduction of exogenous genes into cultured cells using DEAE-dextran-MMA graft copolymer as non-viral gene carrier

Yuki Eshita et al. Molecules. .

Abstract

Comparative investigations were carried out regarding the efficiency of introduction of exogenous genes into cultured cells using a cationic polysaccharide DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextran-methyl methacrylate graft copolymer; DDMC) as a nonviral carrier for gene introduction. The results confirmed that the gene introduction efficiency was improved with DDMC relative to DEAE-dextran. Comparative investigations were carried out using various concentrations of DDMC and DNA in the introduction of DNA encoding luciferase (pGL3 control vector; Promega) into COS-7 cells derived from African green monkey kidney cells. The complex formation reaction is thought to be directly proportional to the transformation rate, but the complex formation reaction between DDMC and DNA is significantly influenced by hydrophobic bonding strength along with hydrogen bonding strength and Coulomb forces due to the hydrophobicity of the grafted MMA sections. It is thought that the reaction is a Michaelis-Menten type complex formation reaction described by the following equation: Complex amount = K1 (DNA concentration)(DDMC concentration). In support of this equation, it was confirmed that the amount of formed complex was proportional to the RLU value.

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Figures

Figure 1
Figure 1
Transfection of COS-7 cells with sample 1 of DEAE-dextran and DEAE-dextran-MMA graft copolymer. The grafting rate is 130% for sample 2 at 10 mg/mL, sample 3 at 20 mg/mL, and sample 4 at 28.6 mg/mL.
Figure 2
Figure 2
DNase degradation times for foreign DNA complex with DEAE-dextran-MMA graft copolymer and DEAE-dextran, respectively. DNase I degrades both double-stranded and single-stranded DNA endonucleolytically, producing 3´-OH oligonucleotides. Toluidine Blue (TB) is isolated in water from DNA after the degradation, as the DNA is stained with TB. This shows the absorbance of TB isolated from DNA in each sample in the water with a spectrophotometer.
Figure 3
Figure 3
Transfection of COS-7 cells with samples of DEAE-dextran and DEAE-dextran-MMA graft copolymer having a grafting rate of 130% and including 0.075 μg of DNA. Maximum luciferase expression within each experiment was set at 100%.
Figure 4
Figure 4
Infra-Red absorption spectra: a, complex of DDMC/DNA; b, DDMC; c, complex of DEAE-dextran/DNA; d, DEAE-dextran.
Figure 5
Figure 5
Schematic drawing of putative delivery pathways for foreign DNA complex with DEAE-dextran-MMA graft copolymer.
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