Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination.
We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses. A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward. We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants. Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required. We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis. Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.
Peakman
T C
TC
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, UK.
Harris
R A
RA
Gewert
D R
DR
D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D019426:Integrases; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D001616:beta-Galactosidase