crossing daf-2 into daf-5

Hi,
I wish to cross daf-2 mutants into daf-5 mutant background? I was wondering if there is any phenotypic screen that I can do to make the double mutants. Does loss of daf-5 suppress dauer formation of daf-2 at 25C?
Thanks

Hi Joan. daf-2 and daf-5 are in two different dauer formation pathways. You probablly won’t see the great suppression of daf-5/daf-8 in this daf-5/daf-2 situation.
In general, a phenotype that does not show 100% is not a good marker for outcrossing. I would suggest just to use the generic PCR protocol (genotype screening) to outcross this two strains.
Hope this helps.

It doesn’t sound like joan3011 is looking for outcrossing advice, but rather advice on constructing the double mutant. Also, daf-5 mutants are Daf-d, while daf-8 mutants are Daf-c.

Mutations in daf-2 confer a strong temperature sensitive Daf-c (constitutive) phenotype, while mutations in daf-5 confer a Daf-d (defective) phenotype. Mutant daf-5 will not suppress mutant daf-2, unless you use some funny alleles and come up with a surprise result. Also, you never want to build doubles in a pathway based on teh assumption of suppression: this is a great way to end up with a strain that is not what you think it is, and is the source of some spectacular historical errors in pathway model building. I won’t name any names.

Daf-ds are hard to track based on this phenotype, and that is definitely true for daf-5. So I would use a balancer. daf-5 is located way out on IIR. mnC1 looks like it balances. But is there a reason you want daf-5 and not daf-3 mutations? daf-3 is on X and I believe one of the alleles is a deletion, and so trackable by PCR.

Anyway, I would cross daf-2(e1370) het males to a strain with het mnC1, then cross the resulting males (~25 of them) to your daf-5 allele of choice (4 L4s) and pick ~40 L4 progeny to grow at 25C. This will work if your cross was very efficient, denoted by the presence of ~50% males on the cross plate. Find plates that throw both dauers and the DpyUnc of mnC1: from that plate, pick ~20 dauers singly to plates at 15C, recover them, and see which plates no longer throw the DpyUnc. You selected for daf-2 homozygotes, and you ensured the presence of your daf-5 mutation by going with plates where the balancer was lost. Voila!

The numbers game is critical. If you have a very efficient cross into daf-5, you should see that 50% males. Pick L4 herms, 32 minimum (but in a cross like this I’d typically go with 40)

Revised version: there is a GFP-tagged mnC1 at the CGC (CGC43, with umnIs32 inserted into the mnC1 chromosome. There is also an older nIs190, but it looks like it can be lost through recombination at a low rate.). If you aren’t in a rush, I would go with CGC43 to make the numbers game more in your favor, and unambiguously identify cross progeny. Also, start with mnC1g het males into daf-2 rather than the way I described it, then pick green on down the line.

May the odds be ever in your favor! :wink:

excerpt:
“Also, you never want to build doubles in a pathway based on teh assumption of suppression: this is a great way to end up with a strain that is not what you think it is, and is the source of some spectacular historical errors in pathway model building. I won’t name any names.”

You don’t have to name names, but I would love to hear more about this, even in general outline. How can we learn from the mistakes from others if we don’t ever hear about them? I was in a lab once that fractionated cytosol to identify a factor that stimulated an in vitro assay. They got it all the way down to the final fraction only to determine that the molecule was the detergent they added to originally lyse the cells, and obviously not some protein factor they were looking for. Always test whether adding a protease kills your activity. Otherwise it might be a small molecule, which could be interesting, or it could be an artefact!

k.

Too coy?

Approach: cross two mutants together, infer epistatic relationship from what segregates. Believe it or not, its been done and published prominently.

But I try not to be negative, and more info would definitely name names. But I will communicate by pm, including references.