Hi All,
Please am having some doubts about the generalization of the life cycle of the different strains of C. elegans that am working with. Cultivating both set of strains under the same conditions with OP50 and at the same temperature (20 degrees Celsius), I noticed that the wild type animals produce eggs a day earlier than the RB strain of animals. From this observation I am thinking the life cycle that is mostly available online is only applicable to the wild type animals, it’s not the same for the RB strain animals. Please can anyone who has observed similar results share his or her own experience or do I still assume that the life cycle of the two strains are the same? And if anyone has a link that describes the life cycle of the RB strain of the animal, please do share. Thank you for your help.
The lifecycle times reported in standard resources like Wormbook and WormAtlas are indeed specific to WT animals at specific temperatures, grown on OP50. Looking at the literature, RB864 looks like some kind of deletion mutant in xpa-1, so nucleotide excision repair is defective. This could certainly explain changes in development. Also, the CGC version of the strain is not outcrossed. Are you working with this strain, or has it been outcrossed? You should never assume that the developmental timecourse of a mutant strain is the same as WT; you should always test this experimentally.
Since it seems like you may have some developmental delay in RB864 (if this is indeed something that interests you), you should
-outcross the strain
-check broodsize and developmental time in RB864 and another xpa-1(lf) strain (either mutant or RNAi)
Thank you so much for your response JordanWard. I am working on the RB864 strain from CGC that was not out crossed. So when you advise that I should outcross this strain, how do I do this? Do you mean I should put maybe N2 strain animals and RB strain of animals on the same plates and see the outcome? Please this is the first time I would be attempting such experiment. Also, I guess while monitoring the RB strain, I should also be looking out for the same features that tend to distinguish the different stages of the wild type strain. For example the L4 stage of the animal which is characterized by the light patch along the body of the animal.
What I mean is that you need to repeatedly cross into a WT background and keep recovering the xpa-1 deletion. This will mean that you should have the xpa-1 mutation in a pretty much WT background (aside from any linked mutations).
You can probably find some resources online, but I cross an N2 male to a strain of interest (RB864 in your case). Then cross F1 males to an N2 hermaphrodite. Repeat this step. Each time you cross to WT is one backcross; you want to do this six times. I typically do three backcrosses, then plate out 12-24 L4s. Genotype these to recover the mutation of interest (should be genotyping primers for the RB864 available at the CGC site: http://www.cgc.cbs.umn.edu/strain.php?id=11642)
Homozygose the mutation and repeat for another three backcrosses. And yes, use your knowledge of N2 to stage your mutant (unless there are obvious phenotypes).
Thank you JordanWard. I would put your advice into practice.