Abstract
To create a system of yeast FLP recombinase-mediated recombination in the silkworm, Bombyx mori, we constructed plasmids for an extrachromosomal excision assay, a helper for producing FLP recombinase and an indicator for the detection of excision. The helper involves an open reading frame coding FLP recombinase and the indicator consists of a β-galactosidase gene interrupted with a neomycin-resistant gene cassette flanked at both ends with the two FLP recombinase target (FRT) sites. To examine whether FLP recombinase works in the silkworm, these plasmids were transfected into cultured cells. Strong β-galactosidase activity was observed when the cells were transfected with positive control plasmid, but no activity was observed when indicator alone was used. Weak activity was observed when the indicator was co-transfected with the helper. Cytochemical staining of these transfected cells gave data supporting this result. The same result was obtained when these plasmids were injected in the eggs of the silkworm and stained with X-Gal. Southern blot analysis showed that the DNA fragment caused by the recombination was detected in the DNA extracted from the eggs injected with both indicator and helper, indicating that FLP recombinase works in silkworm cultured cells and embryos, and suggesting that the constructs we made are useful for application of the FLP recombinase to genetic manipulation experiments with silkworms.
