How to integrate?

As mentioned above, there are multiple ways to use the PhiC31 system to make transgenics. Here, we will describe the methods used in our labs. 

We use genomic landing platforms containing 221 bp attP integration sites and integration vectors containing 285 bp attB sites.

The highest integration frequencies have been achieved using lines homozygous for both the landing platform and the integrase source. Worth mentioning, this is clearly the most convenient way to insert a transgene. Using these lines, one simply has to collect embryos from these flies and inject a suitable integration vector. Recently we established a larger set of such doubly homozygous lines, and almost all of them carry a codon-optimized integrase on the X chromosome. Having the integrase on the X allows for quick removal of the integrase after injection.

If a doubly homozygous line does not exist for a desired landing platform, one can either construct the line or simply inject the progeny of a cross. In this case, the direction of the cross is somewhat important, depending on the specific integrase line used. Our integrase sources are driven by the vasa or nanos promoters. Although vasa has quite high zygotic expression in the germline, nanos is mostly delivered as a maternal product. To take advantage of this maternal expression it is preferable to cross nanos-PhiC31 females homozygous for the integrase source to males that are homozygous for the landing platform (though even with the nanos driven sources, rare integration can be seen doing the crosses in the opposite direction). In the case of a vasa-driven integrase the direction of the cross is not so relevant.