I have a homozygous mutant for a C. elegans gene of interest and outcrossed 8 L3/L4 larvae of this mutant with 12 him-5 males to clean the genetic background. After confirming that they have mated (by noting high frequency of males in the F1 progeny), I picked 8 F1 males and put them to a new plate with 12 N2 hermaphrodites. I also got lots of males in the F2 progeny, suggesting the cross has also worked this time. I isolated 24 of these F2 worms onto single plates and did SW PCR, hoping to get the mutation in some of them, using appropriate primers.
But when I do this, I get no bands. So far, I have set up three such crosses with the mutants of interest. If somebody could share advice, tips, suggestions or troubleshoot the problem from their experience and greater knowledge on this matter, I would be very grateful. This cross is the very first step of my research thesis, and so is crucial for me.
the problem seems to be the SW PCR, did you get a band with the wt or your original mutant?
First be sure, that the PCRis working before starting the cross.
Check all parameters of the lysis and the PCR, especially the incubation temperature of the proteinase K.
For me it took half a year to outcross my mutants.
yeah my positive control all worked fine. I am absolutely positive there is nothing wrong with either the lysis with protease K stage or preparing the single worm PCR. The mutation just seems to have vanished from the population - N2 definitely have mated with the F1 (male) progeny because I see plenty of males in the next F2 progeny. Given this, I cannot think of any reason of consistent absent of mutation
I would recommend testing your SW-PCR protocol on individual F1 males. Those must be heterozygous for your mutation, so they should produce both the WT and mutant PCR products. If not, then there’s something wrong with the protocol or experimental design. If so, then set up F1xF1 matings and repeat the SW-PCR on individual F2 males. You should observe WT:heterozygous:mutant genotypes in a 1:2:1 ratio. If the last class is missing, write back and we can discuss possible explanations.
When you say “I isolated 24 of these F2 worms onto single plates and did SW PCR, hoping to get the mutation in some of them, using appropriate primers. But when I do this, I get no bands.” are you talking about males or hermaphrodites?
If you are referring to males it sounds like your mutation is on the X chromosome.
P0: H m/m x M +/0
F1: M m/0 x H +/+
F2: M +/0 (because to be male the animals can not inherit the X chromosome with the mutation, and the X must be wild-type from the hermaphrodite).
I did SW PCR with F2 hermaphrodites only, and not males. So half of the F2 progeny should theoretically be hets for the locus of interest and so should show bands if looking for the mutation with specific primers. It did cross my mind to genotype F1 males to confirm the mutation is there, but since the very existence of (plenty of) males on plate is evidence of outcrossing I believe we can safely assume they are product of mating and are therefore hets (I used the same plate for cross as for the positive control, which did show bands).
One reason I can think of is there had been a random mutation in the N2 that led to increased number of males in the WT and so presence of several males on plate does not necessarily mean they are the product of successful mating between N2 and F1 males. But I checked the N2 plates - they look very normal. One other thing that could have happened is the males I picked for the second cross with N2 were actually the same him-5 males I had used in the first cross and not F1 males - but since I saw plenty of males (almost at 25%), as opposed to 12 him-5 males I had originally put on the plate, I don’t think it a possibility either.
Anyhow I am starting my fourth attempt at this cross…healthy worms from clean plates, no contamination, young males…let’s see what happens. Thanks for all of your help. Please do let me know if you have suggestions.
did you check the positive control in parallel to the F2 worms?
For me the problem was, that sometimes the lysis did not work (I dont know why) and I got no bands.
An other thing is that you should get two bands, one wt and one mutant band (heterozygous).
If it is not a problem of the lysis, you should get at least the wt band.
The rationale for genotyping the F1 males is to test your SW-PCR protocol, not to test if they are heterozygous.
To insure that you’re picking F1 males, transfer the hermaphrodites from the first cross onto new plates after 24hr and use the male progeny for the second cross.
When you’ve tried an experiment multiple times and don’t observe the expected result, it’s most often a problem with the protocol or experimental design. Question your assumptions and rigorously test those possibilities first before invoking some less likely explanation (see Occam’s Razor).
I agree that you need to prove to yourself that your primers can indeed show you the heterozygous animals. If your genotyping scheme is “duplex PCR” using all three primers in your mix to detect a deletion band of the correct size, it doesn’t alway work. Sometimes sets of primers that work 2-by-2 don’t work when you design them so that all three are used together. You might need to do two separate PCR reactions - one to look for the presence or absence of your WT band, AND one to look for the presence or absence of your mutant (deletion) band. Then compare your two gels. Sometimes you can get an idea of whether the duplex PCR is likely to work my making a “fake het” PCR mix using 1ul of N2 lysate and 1ul of mutant lysate. Finally, if you are using duplex PCR, it is essential that your three primers are designed so that the WT band is the smaller PCR product, while the deletion band gives you a larger band. (Note – you must do this so that if you do see what looks like homozygous mutant, you will know that there is not the possibility of it being heterozygous and that you just don’t see the other band because the “smaller product” outcompeted the larger in your PCR.) There is a detailed description of this on Michael Koelle’s website. Look at Part VI of the knockout protocol. Hope this helps.
One problem may arise when some hermaphrodites were not mated to begin with. To increase the chance of getting cross progeny of the desired genotype, I usually set up crosses with one hermaphrodite with ~10 males on each plate. If you put down say 5 hermaphrodites with males, the male progeny you see may be coming from 3 mated hermaphrodite and the other one may not be mated. I don’t think this will be obvious to tell just by the number of male progeny on the plate. This will not be a problem for your first cross, since you’re picking male progeny, but it will be an issue for your second cross. Hope this helps. Good luck!