[Zbrafish] Re: rescuing a known mutation
(by cmoens from fhcrc.org)
Tue Dec 11 14:54:57 EST 2007
This is a hard problem. There are many obvious reasons why it might
not be possible to rescue a phenotype with mRNA injection at the one-
cell stage. Convergent extension genes are an example, where either
loss- or gain-of-function causes the same phenotype. For convergent
extension mutants this hasn't been a problem because typically there
are multiple mutant alleles, which is the very best evidence that you
have cloned the right gene. But for CE morpholino phenotypes rescue
has been difficult in some cases although carefully titrating the
mRNA down sometimes gives rescue - you could try this. You mentioned
that the mRNA causes a "deleterious phenotype". Is it the same as the
loss-of-function phenotype, or something worse? If it involves a lot
of cell death have you considered co-injecting the p53 MO to try to
prevent the death and perhaps detect rescue that way?
There are papers (for instance the original acerebellar cloning
paper) where people used the kind of data you mention - the fact that
a mutant mRNA does not cause the same phenotype as the wild-type mRNA
- as evidence that their missense mutation was deleterious, but I
don't know if that would hold up anymore.
If you have shown that high-resolution mapping points you to your
gene (zero recombinants in many hundred or preferably over a thousand
mutants) and that your phenotype can be phenocopied unambiguously
with morpholinos, this is pretty solid evidence. Better evidence
would be to rescue of course. Have you tried injecting DNA - perhaps
a BAC containing your gene? Although inheritance will be mosaic, at
least your gene will be expressed under its normal regulation. Or you
could try injecting your cDNA in a Tol2 backbone with an inducible
promoter to avoid effects of ectopic expression during gastrulation.
Better evidence still would be a second non-complementing allele.
Have you considered TILLING? That would nail it, but it could be
months before you have the data...
I hope others post responses to your question because even after all
these years the criteria for proof that you have cloned the right
gene are still not the same from journal to journal or from reviewer
Cecilia B. Moens
Howard Hughes Medical Institute
Division of Basic Science
Fred Hutchinson Cancer Research Center
B2-152, 1100 Fairview Ave. N.
Seattle, WA 98109-1024
office: (206) 667-5627
lab: (206) 667-5697
fax: (206) 667-3308
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