<i>TRIM5α</i>/Cyclophilin A-Modified MDBK Cells for Lentiviral-Based Gene Editing
The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin–Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of &l...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-06-01
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| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/17/7/876 |
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| Summary: | The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin–Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of <i>TRIM5α</i>, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a <i>TRIM5α</i> knock-out MDBK-iCas9 cell line MDBK-iCas9<i><sup>TRIM5α−/−</sup></i> without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9<i><sup>TRIM5α−/−</sup></i> displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9<i><sup>TRIM5α−/−</sup></i> and MDBK-WT cell lines, suggesting that <i>CypA</i> functions independently with <i>TRIM5α</i> as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine <i>TRIM5α</i> and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells. |
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| ISSN: | 1999-4915 |