Outcross

Model Organism: Worms Methods & Reagents
1

Hello,

I am new working with c. elegans.

I am supposed to outcross my worm strain OH9330 with a wild-type background to clean it up and to have my ot354 mutation. The problem is that the allele is a single base substitution.

How can I follow my mutation, if I don’t use PCR? The primers that I’ve designed bind in the same way to my mutant and my wild type (N2)

Thank You

2

There’s a few options:

  1. Follow it in trans with a pair of closely linked visible markers; in this case, you could use dpy-10(e128) unc-4(e120). This can be extremely time- and labor-efficient, if (as in this case) suitable linked markers are readily available. This will mean you will not have outcrossed in the interval between the two markers, but in this case that’s less than two map units, and you wouldn’t have done much more to outcross that region in any case. Note that it’s important to make sure in each generation that no recombination has occurred within the interval between the two markers.

  2. Phenotype! There’s a bunch of phenotypes associated with hlh-3 mutants, including with hlh-3(ot354). If any of these are seen with ot354 and you can readily score them, they’re an obvious way to follow ot354. If you’re being pushed to outcross ot354 this may not be your best bet (maybe the phenotype isn’t caused by ot354), though you can always sequence the ot354 site after you’re done outcrossing this way.

  3. Allele-specific PCR. I know you ask for non-PCR alternatives, and in any case I don’t have personal experience doing allele-specific PCR from which to make a concrete suggestion. But if you’re not already, you should be aware that methods for this exist, and some people really like them.

  4. CRISPR. Make a strain carrying the ot354 mutation de novo! Not a terribly serious suggestion (I’d say these four suggestions are basically in descending order of seriousness, with (1) being the most efficient method), but it’s a viable option.

3

Just to add to Hillel’s comment #1: Rather than using genetic markers, you could also use one of the fluorescent markers that we inserted randomly by minimos transposition (see http://www.wormbuilder.org/reagents/fluorescent-marker-strains/ ). They’re bright and spaced at reasonably high density (there’s a Peft-3:GFP:NLS less than 1 map unit away from hlh-3). A dominant, fluorescent marker makes some of the genetics slightly easier since you’ll know when you’ve re-homozygosed your allele by the absence of fluorescence.

The caveat to the genetic/fluorophore approach is that you’ll never clean up your strain more than what you outcross it against…. outcrossing against N2 or making the allele by CRISPR would mostly solve that issue.

4

A couple of responses to Christian’s comments:

  1. The fluorescent marker insertions are a tremendous resource, but they’re not best suited to this application: because they’re dominant, you will have to mate them in and back out again for each outcross, meaning two generations per outcross. On the other hand, if you mate ot354 males to dpy-10 unc-4 hermaphrodites and pick the male progeny (or mate dpy-10 unc-4/+ males to ot354 hermaphrodites, then mate one male per plate from that cross to dpy-10 unc-4 hermaphrodites so you can spot the males that had a dpy-10 unc-4 chromosome), you can in each subsequent generation mate wild-type male progeny to the dpy-10 unc-4 strain, and get one outcross per generation (though homozygosing ot354 at the end of the process will be slightly more work than using fluorescent markers).

  2. The use of paired recessive visible markers, if done right, means you can’t lose the ot354 mutation by recombination and don’t have to sequence your finished outcrossed strain. The use of a single marker like Christian’s integrated fluorescent reporters will mean there’s a slight risk recombination will have occurred, and you should sequence the ot354 site in the finished, outcrossed strain.

  3. Christian is of course correct that outcrossing to a mutant strain means that you’re not so much “cleaning up” your genetic background as you’re replacing it with a different set of background mutations. If this troubles you, you can extensively outcross your marker strain to the wild type before using it. How much you worry about this depends a lot on your reasons for outcrossing. If you fear that your strain’s interesting phenotype might not result from the mutation you know about, outcrossing to a different set of background mutations is a perfectly good way to test this proposition, even though the strain isn’t getting genetically “cleaner”.

5

I’m not sure you guys are answering the real question, which is how to follow a point mutation (at all) or during outcrossing, if not by sequencing all the time.

The good news is, there is what we call a snip-SNP for the ot354 allele. If you go to this website on worm base talking about the ot354 allele, you will be able to compare the mutant to the wild type sequence:

http://www.wormbase.org/species/c_elegans/variation/WBVar00278422#02-45-3

The wild type version with this random 1Kb chunk is cut with DpnI twice, wild the t354 allele is cut just once. As a result, you can PCR the gene from wild type and mutant, cut both with DpnI, and detect either allele or both.

I’ve attached images of a digest using the EnzymeX program, but you can check it for yourself with ApE, etc. I would check what PCR would look like with the oligos you say you already have, and determine whether they can be used to distinguish the two versions. Always run wild-type and mutant controls during outcrossing. Usually the strategy is to use N2 males to cross to your homozygous mutant, all of the males will then be hets (and you don’t have to genotype), then mate those back to N2 hermaphrodites, moving the animals everyday. Pick L4’s progeny from that cross and genotype for the presence of the mutant allele. If it is there, you can homozygose the mutant or keep going with with mating with N2 males. Those males may or may not pick up the mutant, so you will have to set up more females to genotype at the next step to make sure you haven’t lost it. If you’re worried, you can just homozygose at 2x (the female above), and restart the process. The great thing about this approach is that is possible to outcross with the mutant never being homozygosed, so you can really make good time and always have a clean chromosome in trans to the mutant one.

6

kevinem,

It’s a good point that rather than optimizing allele-specific PCR for ot354 it can be scored by restriction digest following PCR. This will however be more labor-intensive and consume more reagents than following ot354 in trans using either visible phenotypic markers or integrated transgenes.

If our recommendations aren’t sufficiently clear for wen to understand, wen can ask for clarification here or can discuss them with their advisor.

7

Thank you! that is very helpfull