Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.
The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.
Groth
Amy C
AC
Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA. calos@stanford.edu
Fish
Matthew
M
Nusse
Roel
R
Calos
Michele P
MP
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D015139:Blotting, Southern; D002478:Cells, Cultured; D017931:DNA Primers; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D016662:Virus Integration / Q000235:genetics
C488991:AttB protein, Drosophila; D017931:DNA Primers; D029721:Drosophila Proteins; D019426:Integrases