Abstract
Background
Currently, to delete an essential gene from a baculovirus genome, a cell line stably expressing the gene to be knocked-out should be first generated, which is time-consuming. Alternatively, essential genes can be deleted in E. coli using the λ Red recombination system, which requires an electroporation system. Here, based on homologous recombination in insect cells, we develop an alternative efficient system that requires neither generation of a cell line nor an electroporation system.
Methods and results
Using puc19-based inverse PCR, a transfer vector for deleting BmNPV orf92 (Bm92, an essential gene) was efficiently constructed. A copy of Bm92 was introduced into the polyhedrin locus of BmNPV bacmid. The transfer vector was then co-transfected into BmN cell with the modified bacmid to enable homologous recombination at the Bm92 locus. An agarose-free approach was developed for the purification of Bm92-disrupted bacmid viruses in insect cells. Subsequently, BmN cells were co-infected with purified Bm92-disrupted bacmid viruses and unmodified bacmid viruses to allow recombination at the Tn7 insertion site between the two viruses. Finally, bacmid DNA extracted from BmN cells was transformed into chemically-treated competent DH10B cells, and blue colonies containing Bm92-disrupted bacmid were selected using PCR.
Conclusions
For its efficiency and convenience, the system has great potential to be used for the generation of baculovirus knockout mutants.
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Acknowledgements
We are grateful to Prof. Shiqing Xu and Prof. Xiaolong Hu for providing the BmN cell line.
Funding
This study was supported by the China Agriculture Research System of MOF and MARA, the grants from National Natural Science Foundation of China (Grant 32172795),the Young Elite Scientists Sponsor ship Program by CAST (Grant 2018QNRC001), the Science & Technology support Program of Suzhou (SNG201925, SNG201912), the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF): CX(21)2039, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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WJS: Conceptualization, Methodology, Experimental design, Writing—original draft. JWQ: Cell culture, plasmid extraction, Writing-review. YYR: Cell culture, plasmid extraction, Writing-review. WBW: Resources, Writing—review & editing. FCL: Resources, Writing—review & editing. BL: Supervision, Project administration, Writing—review & editing.
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Su, W., Qu, J., Ren, Y. et al. A novel system for the generation of baculoviruses mutant for an essential gene. Mol Biol Rep 49, 6443–6452 (2022). https://doi.org/10.1007/s11033-022-07458-2
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DOI: https://doi.org/10.1007/s11033-022-07458-2