how to mate vulvaless & male tail defect strain

Model Organism: Worms Methods & Reagents
1

I am trying to mate sur-2(ku9) mutants to make double mutants. However, this strain is both vulvaless and has an abnormal male tail. So, it is not possible to mate using the hermaphrodite, and so far I have not had any luck mating the males (even into N2 hermaphrodites).
Can anyone suggest a method to improve male mating efficiency of this strain? Double mutants with this allele do exist, so it must be possible…
Also, does anyone know if it is possible to harvest and inject sperm into vulvaless mutants?

Thanks!

2

There are a number of things you could try in this situation. Yes, manual transfer of sperm into a vulvaless animal has been reported (though I can’t find the reference at the moment). You could also try methods that were used to cross Vul strains in the past: looking through very large numbers in the hope of finding an extremely rare animal that can mate, or mutagenizing and screening for suppressors of Vul. Slightly better than that, you could try RNAing a gene with a loss-of-function Muv phenotype in the hope of bypassing your Vul phenotype (though I haven’t looked to see what’s known about such interactions in the case of sur-2).

But in this case, none of these approaches should be necessary. People have successfully worked with sur-2 in the past, and it is more than likely that they saved strains heterozygous for sur-2 in their collections, so that they would have a strain available with which they could make double mutants when needed. There is no such heterozygote at the CGC; I recommend that you contact groups that have published using sur-2(ku9), especially groups that mapped it or made double mutants with it, and ask if they have a heterozygous strain they would be willing to send you.

3

Re: manual transfer of sperm, Craig LaMunyon is, to my knowledge, the only person who has successfully performed this technique (and it wasn’t easy). I second Hillel’s suggestion to contact labs that have worked with the sur-2 strain and ask for the heterozygous lines.

4

Thanks for your suggestions! Unfortunately the sur-2 hets are not available from labs that published on this gene. But I will try RNAi against lin-1 to get Muv…unless it is lethal…

5

One other suggestion: it will be inefficient because of the low brood size of Vul animals, but what about injecting a sur-2 rescuing construct? After all, you know what the mutant gene is.