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Gene delivery in adherent and suspension cells using the combined physical methods

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Abstract

Physical methods are widely utilized to deliver nucleic acids into cells such as electro-transfection or heat shock. An efficient gene electro-transfection requires the best conditions including voltage, the pulse length or number, buffer, incubation time and DNA form. In this study, the delivery of pEGFP-N1 vector into two adherent cell lines (HEK-293 T and COS-7) with the same origin (epithelial cells), and also mouse bone marrow-derived dendritic cells (DCs) was evaluated using electroporation under different conditions alone and along with heat treatment. Our data showed that the highest green fluorescent protein (GFP) expression in HEK-293 T and COS-7 cells was observed in serum-free RPMI cell culture medium as electroporation buffer, voltage (200 V), the pulse number (2), the pulse length (15 ms), the circular form of DNA, and 48 h after electro-transfection. In addition, the highest GFP expression in DCs was detected in serum-free RPMI, voltage (300 V), the pulse number (1), the pulse length (5 ms), and 48 h after electro-transfection. The use of sucrose as electroporation buffer, the pulse number (2), and the pulse length (25 ms) led to further cytotoxicity and lower transfection in HEK293T and COS-7 cells than other conditions. Moreover, the high voltage (700 V) increased the cell cytotoxicity, and decreased electro-transfection efficiency in DCs. On the other hand, the best conditions of electroporation along with heat treatment could significantly augment the transfection efficiency in all the cells. These data will be useful for gene delivery in other cells with the same properties using physical methods.

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Acknowledgements

Authors acknowledge the financial support by Pasteur Institute of Iran for experimental works (Grant No. 1135).

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  1. Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran

    Kimia Kardani, Alireza Milani & Azam Bolhassani

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  1. Kimia Kardani
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Correspondence to Azam Bolhassani.

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10616_2022_524_MOESM1_ESM.tif

Supplementary Figure 1: The pEGFP-N1 plasmid map (A) and agarose gel image of NotI digestion (B). MW is molecular ladder (1 kb, Fermentas). Supplementary file1 (TIF 431 KB)

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Kardani, K., Milani, A. & Bolhassani, A. Gene delivery in adherent and suspension cells using the combined physical methods. Cytotechnology 74, 245–257 (2022). https://doi.org/10.1007/s10616-022-00524-4

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