title: Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification.
abstract: Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.
authors.affiliation: Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany. juergen.stech@fli.bund.de
authors.forename: Jürgen
authors.initials: J
authors.lastname: Stech
authors.forename: Olga
authors.initials: O
authors.lastname: Stech
authors.forename: Astrid
authors.initials: A
authors.lastname: Herwig
authors.forename: Hermann
authors.initials: H
authors.lastname: Altmeppen
authors.forename: Jana
authors.initials: J
authors.lastname: Hundt
authors.forename: Sandra
authors.initials: S
authors.lastname: Gohrbandt
authors.forename: Anne
authors.initials: A
authors.lastname: Kreibich
authors.forename: Siegfried
authors.initials: S
authors.lastname: Weber
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Klenk
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D005814:Genes, Viral; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus