> Hello, people!
>> I have a classical yeast genetics question that I am not sure.
> The problem is I have a heterozygote knock-out diploid cells in h90 background (homothallic strain). {It's a long story why I didn't start with a heterothallic strain.}
> Anyway, this mating type switching causes some potential problem. So, I thought about crossing with heterothalic strain to move the knocked-out allele to the heterothallic background. Then, I got confused.
> h90 cells are h90 leu1-32,ura4D18, ade6-M210/h90 leu1-32,ura4D18,ade6-M210, KanMX6(G418 selection)::my gene.
I'm a little unclear how you are maintaining these cells as diploids. pombe in general
prefers being haploid, and h90 cells are usually haploid. They are simply switching, so t
hat any population contains a mixture of functionally h+ and functionally h- haploids that
can mate. But they will only mate if starved, and immediately sporulate and form haploids again.
In the absence of complementing markers (e.g., ade6-M210 /ade6-M216) you won't have
diploids. The strain you have given us the genotype for would not exist as a diploid since it
doesn't have any complementing markers. Its real genotype is
h90 leu1 ura4 ade6 yfg1::kanMX
I suppose if your gene disruption is in an essential gene, you might be able to maintain this as a
diploid, but I'd wonder how you made it to begin with....
> I want to have cells in the genotype of h90(+) leu1-32, ura4D18, ade6M210/h- leu1-32,ura4D18, ade6M216 KanMX6::my gene. So, I can select the diploids using Ade marker. Then, I would like to isolate haploid cells of h- leu1-32, ura4D18,ade6M216, KanMX6::my gene. My gene is located on 2nd chromosome which mat1,2,3 genes are located. I got more confused at this point whether this can be done or not.
Simply being on the chromosome will not affect segregation unless they are closely linked
on that chromosome. Yeast are highly recombinogenic, and markers on the same
chromosome are usually genetically unlinked. Even if two markers are tightly linked,
you can isolate recombinants (using random spore analysis, which allows you to assay
a large population).
I think you are misunderstanding the nature of the h90. An h90 is just
another allele at the mating type locus, as is h- or h+. The h90
strain can be treated as an h+ or as an h- in crosses, although it is so efficient
at mating with itself that you need to first make a diploid using complementing
markers. That is, if you want to move your gene of interest from an h90 to an h+
background, you cross it to an h+ strain with the opposite ade marker, select
diploids, induce sporulation, and screen for h+ kanMX offspring (this can
be done using random spores). Half the offspring will be h+, and half
h90. SImilarly, you can cross the h90 to an h-, make
a diploid, then sporulate.
More information can be found on our web site.
--
-susan
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The Salk Institute, La Jolla CA
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