Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency
ObjectiveTo construct TIR-domain-containing adaptor-inducing interferon β (TRIF) gene knockout mouse NA cell line and hamster BHK-21 cell line using CRISPR/Cas9 technology, evaluate the proliferative capacity of rabies virus (RABV) in these knockout cell lines, and investigate TRIF function during R...
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South China Agricultural University
2025-09-01
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| Series: | Huanan Nongye Daxue xuebao |
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| Online Access: | https://journal.scau.edu.cn/article/doi/10.7671/j.issn.1001-411X.202502016 |
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| author | Weitao HUANG Jiaqi LI Yao PENG Xiangping YIN Xiaofeng GUO Jun LUO |
| author_facet | Weitao HUANG Jiaqi LI Yao PENG Xiangping YIN Xiaofeng GUO Jun LUO |
| author_sort | Weitao HUANG |
| collection | DOAJ |
| description | ObjectiveTo construct TIR-domain-containing adaptor-inducing interferon β (TRIF) gene knockout mouse NA cell line and hamster BHK-21 cell line using CRISPR/Cas9 technology, evaluate the proliferative capacity of rabies virus (RABV) in these knockout cell lines, and investigate TRIF function during RABV infection. Method First, single guide RNA (sgRNA) was designed, and recombinant plasmids pSpCas9-NA-TRIF-KO and pSpCas9-BHK-21-TRIF-KO were constructed for TRIF gene knockout. After plasmid transfection, positive cells were preliminarily screened using puromycin followed by single-cell subclone isolation through limited dilution. The expanded cell populations were subsequently validated by PCR amplification, sequencing, and Western blot identification, TRIF-knockout NA and BHK-21 cell lines were established. Standard challenge strain CVS-11 and vaccine strain BNSP-SAD of RABV were then inoculated into both knockout and wild-type cells. Viral N protein expression levels were detected by Western blot, and viral titers were determined through TCID50 assay to evaluate rabies virus proliferation efficiency in different cell lines. ResultThe recombinant plasmids pSpCas9-NA-TRIF-KO and pSpCas9-BHK-21-TRIF-KO for TRIF-knockout were successfully constructed, the TRIF-knockout NA and BHK-21 cell lines were screened and identified. Compared with the wild-type cell line, the expressions of N proteins in two TRIF-knockout cell lines inoculated with RABV CVS-11 and BNSP-SAD were obviously elevated after 24 h infection, and the virus titer significantly increased after 24 h infection. ConclusionRABV has superior proliferation capacity in TRIF-knockout cell lines compared to wild-type counterparts, and TRIF may have the ability to inhibit viral replication. The findings not only provide substantial support for further investigation of TRIF function, but also establish novel cellular models for RABV cultivation and research. |
| format | Article |
| id | doaj-art-4bc861318ac54bb59b53207447eae77b |
| institution | DOAJ |
| issn | 1001-411X |
| language | zho |
| publishDate | 2025-09-01 |
| publisher | South China Agricultural University |
| record_format | Article |
| series | Huanan Nongye Daxue xuebao |
| spelling | doaj-art-4bc861318ac54bb59b53207447eae77b2025-08-20T03:02:07ZzhoSouth China Agricultural UniversityHuanan Nongye Daxue xuebao1001-411X2025-09-0146562763610.7671/j.issn.1001-411X.202502016202502016Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiencyWeitao HUANG0Jiaqi LI1Yao PENG2Xiangping YIN3Xiaofeng GUO4Jun LUO5College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, ChinaLanzhou Veterinary Research Institute/State Key Laboratory for Animal Disease Prevention and Control , Chinese Academy of Agricultural Sciences, Lanzhou 730046, ChinaCollege of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, ChinaLanzhou Veterinary Research Institute/State Key Laboratory for Animal Disease Prevention and Control , Chinese Academy of Agricultural Sciences, Lanzhou 730046, ChinaCollege of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, ChinaCollege of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, ChinaObjectiveTo construct TIR-domain-containing adaptor-inducing interferon β (TRIF) gene knockout mouse NA cell line and hamster BHK-21 cell line using CRISPR/Cas9 technology, evaluate the proliferative capacity of rabies virus (RABV) in these knockout cell lines, and investigate TRIF function during RABV infection. Method First, single guide RNA (sgRNA) was designed, and recombinant plasmids pSpCas9-NA-TRIF-KO and pSpCas9-BHK-21-TRIF-KO were constructed for TRIF gene knockout. After plasmid transfection, positive cells were preliminarily screened using puromycin followed by single-cell subclone isolation through limited dilution. The expanded cell populations were subsequently validated by PCR amplification, sequencing, and Western blot identification, TRIF-knockout NA and BHK-21 cell lines were established. Standard challenge strain CVS-11 and vaccine strain BNSP-SAD of RABV were then inoculated into both knockout and wild-type cells. Viral N protein expression levels were detected by Western blot, and viral titers were determined through TCID50 assay to evaluate rabies virus proliferation efficiency in different cell lines. ResultThe recombinant plasmids pSpCas9-NA-TRIF-KO and pSpCas9-BHK-21-TRIF-KO for TRIF-knockout were successfully constructed, the TRIF-knockout NA and BHK-21 cell lines were screened and identified. Compared with the wild-type cell line, the expressions of N proteins in two TRIF-knockout cell lines inoculated with RABV CVS-11 and BNSP-SAD were obviously elevated after 24 h infection, and the virus titer significantly increased after 24 h infection. ConclusionRABV has superior proliferation capacity in TRIF-knockout cell lines compared to wild-type counterparts, and TRIF may have the ability to inhibit viral replication. The findings not only provide substantial support for further investigation of TRIF function, but also establish novel cellular models for RABV cultivation and research.https://journal.scau.edu.cn/article/doi/10.7671/j.issn.1001-411X.202502016crispr/cas9tir-domain-containing adapter-inducing interferon β genegene knockoutrabies virus |
| spellingShingle | Weitao HUANG Jiaqi LI Yao PENG Xiangping YIN Xiaofeng GUO Jun LUO Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency Huanan Nongye Daxue xuebao crispr/cas9 tir-domain-containing adapter-inducing interferon β gene gene knockout rabies virus |
| title | Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency |
| title_full | Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency |
| title_fullStr | Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency |
| title_full_unstemmed | Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency |
| title_short | Construction of TRIF-knockout NA and BHK-21 cell lines and effect on rabies virus proliferation efficiency |
| title_sort | construction of trif knockout na and bhk 21 cell lines and effect on rabies virus proliferation efficiency |
| topic | crispr/cas9 tir-domain-containing adapter-inducing interferon β gene gene knockout rabies virus |
| url | https://journal.scau.edu.cn/article/doi/10.7671/j.issn.1001-411X.202502016 |
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