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. 2024:2801:147-176.
doi: 10.1007/978-1-0716-3842-2_12.

Generation of Connexin-Expressing Stable Cell Pools

Abraham Tettey-Matey  1 , Chiara Di Pietro  1 , Viola Donati  1   2 , Fabio Mammano  1   3 , Daniela Marazziti  4
Affiliations

Generation of Connexin-Expressing Stable Cell Pools

Abraham Tettey-Matey et al. Methods Mol Biol. 2024.

Abstract

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

Keywords: Confocal microscopy; Gap junction channels; Hemichannels; Immunofluorescence; Lentiviral transduction.

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References

    1. Elfgang C, Eckert R, Lichtenberg-Frate H et al (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol 129(3):805–817. https://doi.org/10.1083/jcb.129.3.805 - DOI - PubMed
    1. Beltramello M, Bicego M, Piazza V et al (2003) Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells. Biochem Biophys Res Commun 305(4):1024–1033. https://doi.org/10.1016/s0006-291x(03)00868-4 - DOI - PubMed
    1. Beltramello M, Piazza V, Bukauskas FF et al (2005) Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness. Nat Cell Biol 7(1):63–69. https://doi.org/10.1038/ncb1205 - DOI - PubMed
    1. Vargas AA, Cisterna BA, Saavedra-Leiva F et al (2017) On biophysical properties and sensitivity to gap junction blockers of connexin 39 hemichannels expressed in HeLa cells. Front Physiol 8:38. https://doi.org/10.3389/fphys.2017.00038 - DOI - PubMed - PMC
    1. Choi EJ, Palacios-Prado N, Saez JC et al (2020) Identification of Cx45 as a major component of GJs in HeLa cells. Biomol Ther 10(10). https://doi.org/10.3390/biom10101389

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