Abstract
Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.
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References
Van Oers MM, Pijlman GP, Vlak JM (2015) Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology. J Gen Virol 96:6–23
Luckow VA, Lee SC, Barry GF, Olins PO (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67:4566–4579. https://doi.org/10.1128/jvi.67.8.4566-4579.1993
Pijlman GP, van Schijndel JE, Vlak JM (2003) Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J Gen Virol 84:2669–2678. https://doi.org/10.1099/vir.0.19438-0
Hilton S, Kemp E, Keane G, Winstanley D (2008) A bacmid approach to the genetic manipulation of granuloviruses. J Virol Methods 152:56–62. https://doi.org/10.1016/j.jviromet.2008.05.015
Wang H, Deng F, Pijlman GP et al (2003) Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome. Virus Res 97:57–63. https://doi.org/10.1016/j.virusres.2003.07.001
Rohrmann GF (2019) The AcMNPV genome: gene content, conservation, and function. In: Baculovirus molecular biology, 4th edn. National Center for Biotechnology Information, Bethesda, pp 1–84
Kaba SA, Salcedo AM, Wafula PO et al (2004) Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins. J Virol Methods 122:113–118. https://doi.org/10.1016/j.jviromet.2004.07.006
Olszewski J, Miller LK (1997) A role for baculovirus GP41 in budded virus production. Virology 233:292–301. https://doi.org/10.1006/viro.1997.8612
Warming S (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36–e36. https://doi.org/10.1093/nar/gni035
Westenberg M, Soedling HM, Mann DA et al (2010) Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs. Nucleic Acids Res 38:e166–e166. https://doi.org/10.1093/nar/gkq596
Wang H, Bian X, **a L et al (2014) Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res 42:e37–e37. https://doi.org/10.1093/nar/gkt1339
Muyrers J (1999) Rapid modification of bacterial artificial chromosomes by ET- recombination. Nucleic Acids Res 27:1555–1557. https://doi.org/10.1093/nar/27.6.1555
Zhang X, He A, Zong Y et al (2023) Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome. Front Microbiol 14. https://doi.org/10.3389/fmicb.2023.1171500
Pijlman GP, Grose C, Hick TAHH et al (2020) Relocation of the attTn7 transgene insertion site in bacmid DNA enhances baculovirus genome stability and recombinant protein expression in insect cells. Viruses 12:1448. https://doi.org/10.3390/v12121448
Stavropoulos TA, Strathdee CA (2001) Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors. Genomics 72:99–104. https://doi.org/10.1006/geno.2000.6481
Galibert L, Savy A, Dickx Y et al (2018) Origins of truncated supplementary capsid proteins in rAAV8 vectors produced with the baculovirus system. PLoS One 13:e0207414. https://doi.org/10.1371/journal.pone.0207414
Lee HH, Miller LK (1979) Isolation, complementation, and initial characterization of temperature-sensitive mutants of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol 31:240–252. https://doi.org/10.1128/jvi.31.1.240-252.1979
Motohashi T, Shimojima T, Fukagawa T et al (2005) Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochem Biophys Res Commun 326:564–569. https://doi.org/10.1016/j.bbrc.2004.11.060
Pijlman GP, Dortmans JCFM, Vermeesch AMG et al (2002) Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses. J Virol 76:5605–5611. https://doi.org/10.1128/jvi.76.11.5605-5611.2002
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci 97:6640–6645. https://doi.org/10.1073/pnas.120163297
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de Jong, L.A., van Oosten, L., Pijlman, G.P. (2024). Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli. In: Cox, M.M. (eds) Baculovirus. Methods in Molecular Biology, vol 2829. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3961-0_8
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DOI: https://doi.org/10.1007/978-1-0716-3961-0_8
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