This is probably a very easy answer for some of you.
I want to cross a GFP marker (juIs76, unc-25p::gfp) with a mutant. Both are on Chr II. In order to get animals homozygous for my mutant and the GFP I have to choose progeny that are the result of a single recombination event right (or is it double)? I know the likelihood of getting the progeny that I want is dependent on the distance between them (and other factors). The location of jusIs76 is not known. Practically speaking, is this worth trying to do? If so, how many progeny should I screen through? Would you take this approach? Another option is to use another marker that is available for GABAergic neurons that is not on Chr II. I really need something with bright and reliable fluorescence. Any suggestions?
As you say, it all depends on the location on the chromosome of juIs76, including that there’s a very good chance juIs76 may suppress recombination on part of LGII - it was generated by genomic rearrangement, and this is a common problem with such transgenes. It’s worth a try, because it’s easy, but don’t be surprised if it doesn’t readily work. You’ll probably have to get an animal heterozygous for the recombinant chromosome first (you’re less likely to get a double-homozygote instantly than if linkage weren’t involved).
As to other GABAergic reporters, I have no experience using them and so can’t recommend any but it’s really easy to find candidates on WormBase, such as EG1285, containing a translational unc-47::gfp reporter integrated on LGX.
Thank you for the response. What do you mean by “You’ll probably have to get an animal heterozygous for the recombinant chromosome first”? How do I do that?
I was responding to this:
In order to get animals homozygous for my mutant and the GFP I have to choose progeny that are the result of a single recombination event right (or is it double)
My meaning was just that rather than hope to get two recombinant chromosomes simultaneously you’d do better to arrange to look for the single recombinant chromosome you want and then homozygose it. You could for example mate mutant
males into mutant
homozygous hermaphrodites and look for phenotypically mutant
-homozygous progeny expressing gfp
Thanks for your help on this. I sifted through a hundred or so mutant homozygotes and found a GFP positive animal, now laying GFP positive progeny. Thanks so much for your help. This has turned out to be better, actually, because my mutant chromosome has a fluorescent marker linked to it now!