Setting up crosses
From Wikionchus
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Reasons for setting up a cross
The following experiments involve cross-fertilization:
- mapping a mutant
- backcrossing a mutant
- making a double/triple/quadruple mutant
- testing inter-strain fertility as a criterion of membership to a particular species
Some considerations
Before grabbing your pick , be aware of the following facts:
- P. pacificus is a self-fertilizing hermaphrodite with very rare occurrence of spontaneous males
- Hermaphrodites have two X Chromosomes. Males have one X chromosome.
- Even in the presence of males, some offspring will usually be self-fertilized. Consider using a morphologically marked hermaphrodite to distinguish the two.
- Progeny from self-fertilization will be >99% genotypically XX and phenotypically hermaphrodite. Progeny from out-crossing is expected to be 50%hermaphrodite(XX) and 50 % male(X0)
- The X-chromosome in males is always contributed by the hermaphrodite!
Doing the cross
To cross Pristionchus pacificus you first of all need males. If you are unsure whether the strain you are working with produces males check the Pristionchus strain list. If you want to be sure that a particular hermaphrodite has not been mated by a male of an unwanted genotype (from the stock plate it comes from) you should pick L4 virgin hermaphrodites. They do not yet have an vulval opening and are therefore virgins. Before setting up the actual cross it is highly recommended to transfer single animals to a fresh OP50 plate and leave them crawling around for at least 20 minutes. Eggs and small larvae that might stick to the worms can fall off. To cross worms simply place 3-5 L4 hermaphrodites and 3-5 (preferrably yound and "sexy") males of the desired genotypes onto an empty NGM-Agar plate that has been manually (with a pick) been spotted with a little bit of OP50. The worms stay in these little spots of food and (as with humans) the close vicinity is good for the sexual mood the worms are in. (And no, I will not pay 2 Euros for that. Strahlenkranz 19:24, 14 March 2006 (CET)) After at 2-3 days kill the males to avoid them mating to outcross progeny. That would (potentially and depending on what you are trying to do) confuse your later genetic anlysis.
Links
- Back to Pristionchus pacificus Protocols page
