title: Improved universal cloning of influenza A virus genes by LacZα-mediated blue/white selection.
abstract: Reverse genetics of influenza A viruses facilitates both basic research and vaccine development. However, efficient cloning of virus gene segments was cumbersome in established systems due to the necessary cleavage of amplicons with outside cutter restriction enzymes followed by ligation. Occasionally, virus genes may contain cleavage sites for those enzymes. To circumvent that problem, we previously established target-primed plasmid amplification using the negative selection marker ccdB cloned into the plasmid pHW2000, flanked by the highly conserved gene segment termini. Here, we further introduced the LacZα fragment downstream of the ccdB region for additional ad-hoc selection of transformed bacteria by blue/white pre-screening. For comparison, we cloned three gene segments (PA, HA, and NS) from the influenza strain A/Swine/Belgium/1/1979 (H1N1) (SwBelg79) into plasmid vectors pHWSccdB and pHWSccdB-LacZα and observed same cloning efficiency. Furthermore, the plasmid pHWSccdB-LacZα allows easy elimination of bacterial colonies containing empty plasmid clones. Using this improved plasmid, we obtained the complete genomic set of eight functional plasmids for SwBelg79.
authors.affiliation: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald - Insel Riems, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Wessels
authors.affiliation: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald - Insel Riems, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Stech
authors.affiliation: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald - Insel Riems, Germany.
authors.forename: El-Sayed M
authors.initials: el-SM
authors.lastname: Abdelwhab
authors.affiliation: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald - Insel Riems, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Judel
authors.affiliation: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald - Insel Riems, Germany.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
authors.affiliation: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald - Insel Riems, Germany. Electronic address: juergen.stech@fli.bund.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Stech
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D005820:Genetic Testing; D005822:Genetic Vectors; D009980:Influenza A virus / Q000235:genetics*; D010957:Plasmids; D059386:Reverse Genetics / Q000379:methods*; D012641:Selection, Genetic; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
chemical_list: D001616:beta-Galactosidase
keywords: Influenza A virus; LacZα; Reverse genetics; Universal cloning