New Methods and Technologies J Mol Genet Med (December 2009), 3(2), 225-231 doi: jmgm Published online: 01 December 2009 Full Text: (html | pdf ~610kb | refs) Improved and simplified recombineering approach for influenza virus reverse genetics Qinfang Liu †, Shuai Wang †, Guangpeng Ma †, Juan Pu †, Nicole E Forbes ‡, Eearl G Brown ‡* and Jin-Hua Liu †‡¥* † Laboratory of Animal Infectious Diseases, College of Veterinary Medicine, China Agricultural University, Beijing, 100094 PR China ‡ Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario, Canada K1H 8M5 ¥ The Shandong Animal Disease Control Center, Jinan 250022, Shandong province, PR China *Correspondence to: Jin-Hua Liu, Email: ljh@cau.edu.cn, Tel: +86 10 62733837, Fax: +86 10 62733837; Earl Brown, Email: Earl.Brown@uottawa.ca, Tel: +613 5625800; Fax: +613 5625452 Received: 05 March 2009, Revised: 05 May 2009, Accepted: 20 May 2009 © Copyright The Authors ----------------------------------------------------------------------------------------------------------------- ABSTRACT Typical reverse genetics systems for generating influenza viruses require the insertion of each genome segments by DNA ligation into vectors for genome synthesis and expression. Herein is described the construction and use of a novel pair of plasmid vectors for cloning all eight genome segments of influenza A virus by homologous recombination for influenza virus reverse genetics. Plasmids, pLLBA and pLLBG, were constructed to possess opposing RNA polymerase I and RNA polymerase II transcription units for generating influenza genomic and messenger RNAs, respectively. In addition these promoters flanked a recombination cassette which comprised the conserved 5’ (13bp) and 3’ (12bp) terminal promoters of influenza virus. These vectors differed due to the presence of an A or a G (plus sense) to correspond to differences at nucleotide position 4 among negative-sense influenza virus promoters. The cloning approach involved homologous recombination of each influenza gene segment and the appropriate linearized pLLBA or pLLBG vectors in E. coli. Direct cloning by recombination was simpler and fast er than conventional restriction digestion and ligation methods. This new vector system was successfully used to clone and rescue various influenza viruses and thus has the potential to promote the rapid analysis and vaccine development of novel influenza strains. Keyswords: Influenza A virus, reverse genetics, vector construction, homologous recombination ----------------------------------------------------------------------------------------------------------------- Privacy | Disclaimer |