Optimization of A549 cell transfection efficiency with a plasmid encoding the N-protein of the SARS-COV-2 virus

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To test new antiviral drugs aimed at degrading the nucleocapsid protein (N-protein) of the SARS-CoV-2 virus, it is desirable to have cells expressing the N-protein, for which it is necessary to find conditions for the maximum achievable efficiency of cell transfection with a plasmid encoding this protein. For transfection, polyplexes were used consisting of a plasmid encoding the N-protein fused with the mRuby3 fluorescent protein and polyethyleneimine (PEI)-polyethylene glycol (PEG)-TAT peptide block copolymers. The dependence of the transfection efficiency of human lung adenocarcinoma A549 cells on the PEG/PEI and N/P ratios (the ratio of nitrogen in PEI to phosphate in DNA) was studied. Significant positive correlations were shown between transfection efficiency determined by flow cytometry, the N/P ratio, and the proportion of polyplexes sized 40–54 nm. The data obtained can serve as a basis for creating an animal model of lung cells transiently expressing the N protein of the SARS-CoV-2 virus.

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Sobre autores

Y. Khramtsov

Institute of Gene Biology, RAS

Autor responsável pela correspondência
Email: alsobolev@yandex.ru
Rússia, Moscow

T. Lupanova

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru
Rússia, Moscow

A. Rosenkranz

Institute of Gene Biology, RAS; Lomonosov Moscow State University Moscow

Email: alsobolev@yandex.ru
Rússia, Moscow; Moscow

G. Georgiev

Institute of Gene Biology, RAS

Email: alsobolev@yandex.ru

Academician of the RAS

Rússia, Moscow

A. Sobolev

Institute of Gene Biology, RAS; Lomonosov Moscow State University Moscow

Email: alsobolev@yandex.ru

Corresponding Member of the RAS

Rússia, Moscow; Moscow

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2. Fig. 1. Dependences of A549 cell transfection efficiency on the N/P ratio (the ratio of nitrogen in PEI to phosphate in DNA) at different PEG/PEI values ​​in polyplexes. Transfection was performed with plasmid DNA encoding the N protein of the SARS-CoV-2 virus fused with the mRuby3 fluorescent protein. The straight line indicates the linear correlation between transfection efficiency and N/P (r = 0.783, p = 0.013). Average values ​​with the corresponding standard error are shown (n = 7–11).

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3. Fig. 2. (a) Dependence of the proportion of polyplexes (the proportion of all polyplexes is taken as 100%) on their hydrodynamic diameter. The proportion of polyplexes with a diameter of 40–54 nm is shown in gray. (b) Dependence of the transfection efficiency of A549 cells (as a percentage of transfected cells from their total number) on the proportion of polyplexes with a diameter of 40–54 nm. The straight line indicates the linear correlation between these parameters (r = 0.796, p = 0.010).

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