Novel cloning method for recombinant adenovirus construction in Escherichia coli.
pAd(vantage) is a rapid cloning system for generating recombinant adenoviruses. The system is based on manipulating the full-length adenovirus genome as a stable plasmid in E. coli using intron-encoded endonucleases. These intron-encoded endonucleases cut their recognition sequences, which range from 15-39 bp, with high specificity. Their unusual long homing sequence makes them rare-cutting and ideal for use as cloning sites. We report how transgenes can easily be cloned directly into the E1 region of an adenoviral plasmid, followed by transfection into a mammalian packaging cell line, to produce homogeneous recombinant viruses without the need for plaque purification.
Souza
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Genzyme Corporation, Framingham, MA, USA.
Armentano
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D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics
C505032:CFTR protein, human; D004274:DNA, Recombinant; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator