I have a mutant strain of zebrafish wherein the mutation is considered to be directly affecting hormonogenesis. My fish do show some phenotypic aberrations due to this but otherwise seem healthy and live as long as wild type siblings. I have performed a couple of ELISA assays to determine whether hormonal products differ from those in wild types and they do indeed. However, one of the processes I am investigating in these mutants appears to have a very different outcome compared to wild types injected with morpholinos in previously published work. I have no experience with morpholinos yet and though I am aware of their off target effects, I am still wondering whether my mutants may be hypomorphic. What could I try to further investigate this?
A null mutation is defined by the complete absence of the protein encoded by your gene of interest or by a point mutation or deletion that completely inhibits the function of the protein. The easiest way to create a null mutant is by deleting the gene entirely. Making specific mutations in the protein, point mutants or deletions, can create a null but sometimes we do not know enough about the protein function to be sure that its activity is completely abrogated. Your phenotype may not be an indication of a null or a hypomorph if you are using indirect readouts such as hormone levels, which could be affect by other proteins as well. In other words, the terms null or hypomorph refers only to your targeted gene and not to any other direct or indirect effects of that gene product.
Kunal,
I am enclosing a paper in which hypomorphic mutant was created, if you have question on generation, you may ask Dr. van Deursen
If you have an specific antibody that works good, you can do a western blot against your protein of interest in WT and Mutant backgrounds. No protein product of the appropriate size in the mutant suggests that your mutant is NULL. If you have no antibody, check for this paper as an alternative.
https://www.ncbi.nlm.nih.gov/pubmed/27548568
Cheers
Dear Kunal Chopra,
to the contradiction of the observed phenotype of your mutation and the described phenotype using morpholino: it is also possible that knockdown of a given gene leads to a stronger phenotype than a null mutation because of genetic compensation mechanisms
https://www.ncbi.nlm.nih.gov/pubmed/26168398
Hi Kunal Chopra,
You can inject the published morpholino in embryos from a het incross. If the morpholino phenotype is real and your mutation is a null, you should see an underrepresentation of mutant embryos/larvae showing you the phenotype. Make sure you titrate the morpholino amount you inject to minimise non-specific effects. If your mutants are more sensitive to the morpholino than their wt siblings, you are likely looking at a hypomorph.
Hope this makes sense.
Best
Zach
HI Kunal, Good to see you here.
I would try a western as suggested above.
However, it is good to keep in mind the difference between a null and a hypomorph. Whereas a null essentially is devoid of the protein coded by the gene in question there can be a scenario where you have a truncated protein due to your mutation and this might work too but not as well as the WT protein thus giving you somewhat decrease in activity of the protein. Also there is another way of defining a genetic hypomorph where you see a phenotype that would be a result of a null mutation but it is not as pronounced (again might be a result of for example truncated protein product).
Good luck with your experiment. (I see you have moved to Manchester)
Thank you everyone for your insights. I think I will try the Western blot then.
Good to see you too Bilal. Hope you are well :)
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