Abstract
We propose a novel method for direct cloning of foreign genes into baculoviruses which avoids the use of bacterial transfer vectors. The foreign gene to be inserted is derived by PCR using appropriate primers each of which contains an additional 50 nt of baculovirus sequence for homologous recombination between the PCR-derived DNA and the baculovirus DNA, thus accomplishing insertion of the foreign gene into the baculovirus. The direct cloning of green fluorescent protein and beta-glucuronidase in different baculovirus loci is described. The method is simple and avoids the use of cumbersome techniques associated with enzymatic treatment and DNA purification.
Full Text
The Full Text of this article is available as a PDF (37.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ayres M. D., Howard S. C., Kuzio J., Lopez-Ferber M., Possee R. D. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology. 1994 Aug 1;202(2):586–605. doi: 10.1006/viro.1994.1380. [DOI] [PubMed] [Google Scholar]
- Belyaev A. S., Hails R. S., Roy P. High-level expression of five foreign genes by a single recombinant baculovirus. Gene. 1995 Apr 24;156(2):229–233. doi: 10.1016/0378-1119(95)00050-g. [DOI] [PubMed] [Google Scholar]
- Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
- Ernst W. J., Grabherr R. M., Katinger H. W. Direct cloning into the Autographa californica nuclear polyhedrosis virus for generation of recombinant baculoviruses. Nucleic Acids Res. 1994 Jul 25;22(14):2855–2856. doi: 10.1093/nar/22.14.2855. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Khromykh A. A., Meka H., Westaway E. G. Preparation of recombinant baculovirus by transfection of a ligated cDNA fragment without prior plasmid amplification in E. coli. Biotechniques. 1995 Sep;19(3):356–360. [PubMed] [Google Scholar]
- Kitts P. A., Ayres M. D., Possee R. D. Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Res. 1990 Oct 11;18(19):5667–5672. doi: 10.1093/nar/18.19.5667. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kitts P. A., Possee R. D. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques. 1993 May;14(5):810–817. [PubMed] [Google Scholar]
- Lu A., Miller L. K. Generation of recombinant baculoviruses by direct cloning. Biotechniques. 1996 Jul;21(1):63–68. doi: 10.2144/96211bm14. [DOI] [PubMed] [Google Scholar]
- Weyer U., Possee R. D. Analysis of the promoter of the Autographa californica nuclear polyhedrosis virus p10 gene. J Gen Virol. 1989 Jan;70(Pt 1):203–208. doi: 10.1099/0022-1317-70-1-203. [DOI] [PubMed] [Google Scholar]
