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Fast and efficient generation of recombinant baculoviruses by in vitro transposition
- Source :
- Applied Microbiology and Biotechnology. 96:1353-1360
- Publication Year :
- 2012
- Publisher :
- Springer Science and Business Media LLC, 2012.
-
Abstract
- A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.
- Subjects :
- Genetics, Microbial
Baculoviridae
Insecta
Bacillus amyloliquefaciens
Green Fluorescent Proteins
Bacillus
Recombinant virus
Applied Microbiology and Biotechnology
Cell Line
law.invention
Transposition (music)
Ribonucleases
Genes, Reporter
law
Animals
Molecular Biology
Gene
Recombination, Genetic
Barnase
biology
General Medicine
biology.organism_classification
Molecular biology
Cell culture
DNA, Viral
Recombinant DNA
biology.protein
Biotechnology
Subjects
Details
- ISSN :
- 14320614 and 01757598
- Volume :
- 96
- Database :
- OpenAIRE
- Journal :
- Applied Microbiology and Biotechnology
- Accession number :
- edsair.doi.dedup.....cd31cfbcfa3fa3a462ef5b8bbe0a9e5b