Construction of Transgenic Drosophila by Using the Site-Specific Integrase From Phage φC31

The φC31 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. φC31 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 φC31 integrase system and will likely benefit research in Drosophila and other insects.

Phage genetic sites involved in lambda growth inhibition by the Escherichia coli rap mutant.

The rap mutation of Escherichia coli prevents the growth of bacteriophage lambda. We have isolated phage mutants that compensate for the host deficiency. The mutations, named bar, were genetically located to three different loci of the lambda genome: barI in the attP site, barII in the cIII ea10 region, and barIII within or very near the imm434 region. The level of lambda leftward transcription correlates with rap exclusion. Phage lambda mutants partially defective in the pL promoter or in pL-transcript antitermination showed a Bar- phenotype. Conversely, mutants constitutive for transcription from the pI or pL promoters were excluded more stringently by rap bacteria. We conclude that rap exclusion depends on the magnitude of transcription through the wild type bar loci in the phage genome.