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J Virol Methods. 2015 Sep 22. pii: S0166-0934(15)00323-7. doi: 10.1016/j.jviromet.2015.09.009. [Epub ahead of print]
Improved universal cloning of influenza A virus genes by LacZα-mediated blue/white selection.
Wessels U1, Stech O1, Abdelwhab EM1, Judel A1, Mettenleiter TC1, Stech J2.
Author information
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.
Copyright ? 2015. Published by Elsevier B.V.
KEYWORDS:
Influenza A virus; LacZα; Reverse genetics; Universal cloning
PMID: 26404948 [PubMed - as supplied by publisher]
Improved universal cloning of influenza A virus genes by LacZα-mediated blue/white selection.
Wessels U1, Stech O1, Abdelwhab EM1, Judel A1, Mettenleiter TC1, Stech J2.
Author information
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.
Copyright ? 2015. Published by Elsevier B.V.
KEYWORDS:
Influenza A virus; LacZα; Reverse genetics; Universal cloning
PMID: 26404948 [PubMed - as supplied by publisher]