Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines

Abstract

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Keywords: gene-delivery; plasmid-derived protein expression; poly(N-vinylacetamide-co-acrylic acid) bearing d-octaarginine; stable transgene expression; transient transgene expression.

Fig. 1.

Chemical structure of VP-R8. Sourced from [14].

Fig. 2.

Transient green fluorescent protein (GFP) expression in mouse cell lines transfected with pCMV-GFPHA using VP-R8. (a–c) GFP fluorescence (GFP) and bright field (BF) images of C8-D30 (a), RAW264.7 (b), and J774.1 (c) cells that were transfected with pCMV-GFPHA using VP-R8, wherein cells were incubated with the plasmid/VP-R8 mixture for 4 or 24 hr. GFP and BF images of C8-D30, RAW264.7, and J774.1 cells that were or were not transfected with the plasmid by using the PTR are also shown (PTR or non-TF, respectively). The data shown are representative of three (C8-D30), six (RAW264.7), or one (J774.1) independent experiments. The frequency of GFP-positive cells, as measured by flow cytometry, shown as a bar graph. The data have been expressed in terms of mean ± standard deviation (SD; n=3–6). **P<0.01; ****P<0.0001. The data shown are representative of two (C8-D30, [a]), four (RAW264.7, [b]), or one (J774.1, [c]) independent experiments.

Fig. 3.

Transient green fluorescent protein (GFP) expression in human cell lines transfected with pCMV-GFPHA using VP-R8. (a–c) GFP fluorescence (GFP) and bright field (BF) images of HEK293 (a), HeLa (b), and HepG2 (c) cells that were transfected as described in the Fig. 2 legend. The data shown are representative of four (HEK293 and HeLa) or two (HepG2) independent experiments. The frequency of GFP-positive cells, as measured by flow cytometry, shown as a bar graph. The data have been expressed in terms of mean ± standard deviation (n=3). ***P<0.001; ****P<0.0001. The data shown are representative of two (HEK293, [a]) or three (HeLa, [b], and HepG2, [c]) independent experiments.

Fig. 4.

Stable green fluorescent protein (GFP) expression in mouse cell lines transfected with pCMV-GFPHA using VP-R8. (a and b) Top: GFP fluorescence (GFP) and bright field (BF) images of drug-resistant C8-D30 (a) and RAW264.7 (b) cells after selection with G418. Cells were transfected with pCMV-GFPHA using VP-R8, wherein cells were incubated with the plasmid/VP-R8 mixture for 4 or 24 hr, followed by drug selection 3 days after the transfection. GFP and BF images of C8-D30 cells after drug selection are also shown; these cells had been transfected with the plasmid using the PTR. The data shown are representative of three (C8-D30) or four (RAW264.7) independent experiments. Bottom left: crystal violet staining of drug-resistant C8-D30 (a) and RAW264.7 (b) cells after drug selection. The data shown are representative of five independent experiments. Bottom right: The median GFP fluorescence intensity expressed in drug-resistant C8-D30 (a) and RAW264.7 (b) cells after selection, as measured by flow cytometry. The data have been expressed in terms of mean ± standard deviation (n≥13) of three (C8-D30) or four (RAW264.7) independent experiments. *P<0.05; **P<0.01.

Fig. 5.

Stable green fluorescent protein (GFP) expression in human cell lines transfected with pCMV-GFPHA using VP-R8. (a–c) Top: GFP fluorescence (GFP) and bright field (BF) images of drug-resistant HEK293 (a), HeLa (b), and HepG2 (c) cells that were transfected followed by drug selection as described in the Fig. 4 legend. The data shown are representative of three (HEK293 and HeLa) or two (HepG2) independent experiments. Bottom left: crystal violet staining of drug-resistant HEK293 (a), HeLa (b), and HepG2 (c) cells after selection. The data shown are representative of three (HEK293) or two (HeLa and HepG2) independent experiments. Bottom right: The median GFP fluorescence intensity in drug-resistant HEK293 (a), HeLa (b), and HepG2 (c) cells after selection, as measured by flow cytometry. The data have been expressed in terms of mean ± standard deviation (n=9–13) of two (HEK293) or three (HeLa and HepG2) independent experiments.