I have 2 transgenic lines of Drosophila. I would like to cross them and get both transgene in the same fly. To start this cross, does it matter which fly is female or male? Or as long as the female is virgin, it does not matter?
As long as none of your genes are X-linked and you use virgins it shouldn't matter and you'd get the same proportion of double homozygotes. You may want to perform a reciprocal cross (https://en.wikipedia.org/wiki/Reciprocal_cross) if you don't know what chromosome these genes are on (though flybase.org is useful too).