Phage integrases and site-specific integration in Drosophila.

摘要

The ability to reliably insert a transgene into a unique sequence in eukaryotic genomes would be useful in gene therapy and genetic engineering. Recombinases are enzymes that have evolved to efficiently recombine specific, relatively short sequences of DNA. Our lab investigated the ability of CRE to mediate recombination into endogenous human sequences, or pseudo sites, which are similar to its wild-type loxP.{09}Experiments showed that the intermolecular integration frequency in a transient plasmid assay between a wild-type loxP and a pseudo lox sequence was quite low (.1% in the best case). This was attributed to the ability of the CRE recombinase to recombine the resulting hybrid sequences, excising out the newly integrated DNA.; Phage integrases mediate recombination between two different sequences of DNA, attP and attB. Once integration has occurred, the hybrid sites cannot be recombined by the integrase without the presence of an additional enzyme, so the integrated DNA is not excised out. Using the serine integrase &phis;C31, integration into an established EBV vector occurred in human tissue culture cells at a frequency up to 7.5%.; In addition to the &phis;C31 integrase, I studied the ability of a newly discovered family member, the A118 integrase, to mediate recombination in the mammalian environment. The integrase can mediate intramolecular recombination at a frequency of 50% in human 293 cells. In addition, the enzyme can recognize and mediate recombination into a number of human pseudo sites. These pseudo sites are between 35% and 52.5% identical to the wild-type site.; Many other research systems may be manipulated by the serine integrases. I have shown that the &phis;C31 integrase works efficiently in both Drosophila tissue culture cells as well as in Drosophila embryos. The frequency of transgenics into an attP fly line was ∼50% of fertile crosses, a great improvement from the current P element system.; Both the &phis;C31 and A118 integrases mediate efficient and specific recombination reactions in a variety of cell types. These enzymes are likely to become useful tools for the genetic manipulations of many organisms, both in vitro and in vivo.