Optimized condition for pei-based transient transfection of lifeact-gfp/nls-mcherry expressing plasmid used as cell barcode for syncytia live cell imaging

https://doi.org/10.22146/teknosains.89479

Dennaya Kumara(1), Hayfa Salsabila Harsan(2), Metta Novianti(3), Dinda Lestari(4), Endah Puji Septisetyani(5*), Pekik Wiji Prasetyaningrum(6), Komang Alit Paramitasari(7), Edy Meiyanto(8)

(1) Faculty of Pharmacy, Universitas Gadjah Mada; Research Center for Genetic Engineering, National Research and Innovation Agency
(2) Faculty of Pharmacy, Universitas Gadjah Mada; Research Center for Genetic Engineering, National Research and Innovation Agency
(3) Faculty of Mathematics and Sciences, Universitas Syiah Kuala; Research Center for Genetic Engineering, National Research and Innovation Agency
(4) Faculty of Mathematics and Sciences, Universitas Syiah Kuala; Research Center for Genetic Engineering, National Research and Innovation Agency
(5) Research Center for Genetic Engineering, National Research and Innovation Agency
(6) Research Center for Genetic Engineering, National Research and Innovation Agency
(7) Research Center for Genetic Engineering, National Research and Innovation Agency
(8) Faculty of Pharmacy, Universitas Gadjah Mada
(*) Corresponding Author

Abstract


The transfection efficiency positively affects the successful plasmid DNA transfer into cells, with the highlight on the amount of plasmid DNA and its ratio to the transfection reagent. Polyethyleneimine (PEI) is a cost-effective transfection reagent that facilitates DNA transfer by forming positively charged DNA complexes. It allows DNA to interact with negatively charged cell surfaces and enter the cells by endocytosis. In this study, we optimized the condition for transient transfection of life act-GFP/NLS-mCherry-expressing plasmid in BHK-21 and 293T cells using PEI. This plasmid is helpful as a biosensor of the cytoskeleton and nucleus that enables live imaging observation using a fluorescence microscope, for instance, in the observation of syncytium. Here, we optimized two independent variables: the amount of DNA (0.5 and 1 µg) and the ratio of DNA-PEI (1:3 and 1:4). GFP and mCherry expressions were observed at 24, 48, and 72 h post-transfection. As a result, transfection efficiency achieved by using PEI in 293T cells is higher than in BHK-21 cells, which are ~90% and ~50%, respectively. Moreover, amongst four different transfection conditions, in both cell lines, 1 µg of plasmid DNA with a 1:3 DNA-PEI ratio yields the most efficiency with the least amount of toxicity. We used this condition for the syncytia observation in 293T cells as a model of the cell-to-cell transmission of SARS-CoV-2. Syncytia formation was successfully observed by detecting the giant cells expressing GFP/mCherry with multiple nuclei.


Keywords


Lifeact-GFP/NLS-mCherry; polyethyleneimine; SARS-CoV-2; fluorescence protein; transfection efficiency

Full Text:

PDF


References

Bernhofer, M., Goldberg, T., Wolf, S., Ahmed, M., Zaugg, J., Boden, M., & Rost, B. 2018. NLSdb-major update for database of nuclear localization signals and nuclear export signals. Nucleic Acids Research, 46(D1), pp. D503–D508. Available at: https://doi.org/10.1093/nar/gkx1021.

Blackstock, D.J., Goh, A., Shetty, S., Fabozzi, G., Yang, R., Ivleva, V.B., Schwartz, R., & Horwitz, J. 2020. Comprehensive Flow Cytometry Analysis of PEI-Based Transfections for Virus-Like Particle Production. Research: A Science Partner Journal (Washington, D.C.), 2020, p. 1387402. Available at: https://doi.org/10.34133/2020/1387402.

Boussif, O., Lezoualc'h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B., & Behr, J.P. 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America, 92(16), pp. 7297–7301.

Briard, B., Place, D.E., & Kanneganti, T. 2020. DNA Sensing in the Innate Immune Response. 35(2), pp. 112–124.

Chong, Z.X., Yeap, S.K., & Ho, W.Y. 2021. Transfection types, methods and strategies: a technical review. PeerJ, 9, p. e11165. Available at: https://doi.org/10.7717/peerj.11165.

Fus-Kujawa, A., Prus, P., Bajdak-Rusinek, K., Teper, P., Gawron, K., Kowalczuk, A., & Sieron, A.L. 2021. An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro. Frontiers in Bioengineering and Biotechnology, 9. Available at: https://www.frontiersin.org/articles/10.3389/fbioe.2021.701031 (Accessed: 19 April 2023).

He, X., He, Q., Yu, W., Huang, J., Yang, M., Chen, W., & Han, W. 2021. Optimized protocol for high-titer lentivirus production and transduction of primary fibroblasts. Journal of Basic Microbiology, 61(5), pp. 430–442. Available at: https://doi.org/10.1002/jobm.202100008.

Horibe, T., Torisawa, A., Akiyoshi, R., Ohashi, Y.H., Suzuki, H., & Kawakami, K. 2014. Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging. Luminescence, 29(1), pp. 96–100. Available at: https://doi.org/10.1002/bio.2508.

Leroy, H., Han, M., Woottum, M., Bracq, L., Bouchet, J., Xie, M., & Benichou, S. 2020. Virus-Mediated Cell-Cell Fusion. International Journal of Molecular Sciences, 21(24), p. 9644. Available at: https://doi.org/10.3390/ijms21249644.

Liu, H.S., Jan, M.S., Chou, K.C., Chen, H.P., & Ke, N.J. 1999. Is green fluorescent protein toxic to the living cells? Biochemical and Biophysical Research Communications, 260(3), pp. 712–717. Available at: https://doi.org/10.1006/bbrc.1999.0954.

Pandey, A.P. & Sawant, K.K. 2016. Polyethylenimine: A versatile, multifunctional non-viral vector for nucleic acid delivery. Materials Science & Engineering. C, Materials for Biological Applications, 68, pp. 904–918. Available at: https://doi.org/10.1016/j.msec.2016.07.066.

Rajah, M.M., Bernier, A., Buchrieser, J., & Schwartz, O. 2022. The Mechanism and Consequences of SARS-CoV-2 Spike-Mediated Fusion and Syncytia Formation. Journal of Molecular Biology, 434(6), p. 167280. Available at: https://doi.org/10.1016/j.jmb.2021.167280.

Riedl, J., Crevenna, A.H., Yu, J.H., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T.A., Werb, Z., Sixt, M., & Soldner, R.W. 2008. Lifeact: a versatile marker to visualize F-actin. Nature Methods, 5(7), pp. 605–607. Available at: https://doi.org/10.1038/nmeth.1220.

Septisetyani, E.P., Prasetyaningrum, P.W., Anam, K., & Santoso, A. 2021. SARS-CoV-2 Antibody Neutralization Assay Platforms Based on Epitopes Sources: Live Virus, Pseudovirus, and Recombinant S Glycoprotein RBD. Immune Network, 21(6), p. e39. Available at: https://doi.org/10.4110/in.2021.21.e39.

Sergeeva, Y.N., Jung, L., Weill, C., Erbacher, P., Tropel, P., Felix, O., Viville, S., & Decher, G. 2018. Control of the transfection efficiency of human dermal fibroblasts by adjusting the characteristics of jetPEI®/plasmid complexes/polyplexes through the cation/anion ratio. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 550, pp. 193–198. Available at: https://doi.org/10.1016/j.colsurfa.2018.04.035.

Sonawane, N.D., Szoka, F.C., & Verkman, A.S. 2003. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. The Journal of Biological Chemistry, 278(45), pp. 44826–44831. Available at: https://doi.org/10.1074/jbc.M308643200.

Tang, Y., Garson, K., Li, L., & Vanderhyden, B.C. 2015. Optimization of lentiviral vector production using polyethylenimine-mediated transfection. Oncology Letters, 9(1), pp. 55–62. Available at: https://doi.org/10.3892/ol.2014.2684.

Thomas, P. & Smart, T.G. 2005. HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51(3), pp. 187–200. Available at: https://doi.org/10.1016/j.vascn.2004.08.014.

Wünschmann, S. & Stapleton, J.T. 2000. Fluorescence-based quantitative methods for detecting human immunodeficiency virus type 1-induced syncytia. Journal of Clinical Microbiology, 38(8), pp. 3055–3060. Available at: https://doi.org/10.1128/JCM.38.8.3055-3060.2000.

Xie, Q., Xinyong, G., Xianjin, C., & Yayu, W. 2013. PEI/DNA formation affects transient gene expression in suspension Chinese hamster ovary cells via a one-step transfection process. Cytotechnology, 65(2), pp. 263–271. Available at: https://doi.org/10.1007/s10616-012-9483-9.

Zeng, C., Evans, J.P., King, T., Zheng, Y., Oltz, E.M., Whelan, S.P.J., Saif, L.J., Peeples, M.E., & Liu, S. 2022. SARS-CoV-2 spreads through cell-to-cell transmission. Proceedings of the National Academy of Sciences, 119(1), p. e2111400119. Available at: https://doi.org/10.1073/pnas.2111400119.



DOI: https://doi.org/10.22146/teknosains.89479

Article Metrics

Abstract views : 716 | views : 819

Refbacks

  • There are currently no refbacks.




Copyright (c) 2023 Dennaya Kumara, Hayfa Salsabila Harsan, Metta Novianti, Dinda Lestari, Endah Puji Septisetyani, Pekik Wiji Prasetyaningrum, Komang Alit Paramitasari, Edy Meiyanto

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.




Copyright © 2024 Jurnal Teknosains     Submit an Article        Tracking Your Submission


Editorial Policies       Publishing System       Copyright Notice       Site Map       Journal History      Visitor Statistics     Abstracting & Indexing