P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.
We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.
Venken
Koen J T
KJ
Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
He
Yuchun
Y
Hoskins
Roger A
RA
Bellen
Hugo J
HJ
D000818:Animals; D030801:Animals, Genetically Modified; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004260:DNA Repair; D004251:DNA Transposable Elements; D004331:Drosophila melanogaster / Q000235:genetics*; D018014:Gene Transfer Techniques; D017344:Genes, Insect; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011995:Recombination, Genetic; D017906:Siphoviridae / Q000235:genetics*; D019076:Transgenes; D019895:Transposases / Q000378:metabolism
D004251:DNA Transposable Elements; D019895:Transposases