I had a point mutant mouse generated from a company. The mouse has the correct sequence and is a HET. When I set up breeding pairs, only HET and WT pups are born. No homozygous "mutant' pups are though.
The genotyping is determined by RT-PCR and sequencing. There are no homozygous pups in any litter. Any suggestion on what is going on for this?
To find homozygous mutant you have to select a recessive mutant or a female with negative HET, now you will get pups of your own choice.
It is not lethal. We have a patient with this homozygous mutation. And we have confirmed it the pups are not reabsorbed during pregnancy. And our pup numbers are completely normal, so we are not losing them at an embryonic stage.
and all parentals are confirmed heterozygotes by sequencing,of course. No possibility of a het v normal mating.Sorry it had to be asked
Yes, parents were confirmed HETS by sequencing. I really wished that was the problem!
I am sorry Camille I have spent some time trying to work out how to generate a chimaera with the correct genotype in the blood/tissue but not in the sperm/egg and failed . Also thinking that the method of measurement may be at fault...someting like a polymorphism at the 3' end of the primer causing the mutant allele to amplify badly relative to the normal allele. Even considered pcr contamination with the normal allele making all homozygous pcrs look het ( even though I know your negative controls will show this ) and whether there is another sequence in the genome identical to the normal allele . I always found tech help at the companies very helpful so it might be worth calling the company that generated the mutant to see if they ever get this happening while they are developing the mouse line but I am stumped on this one and can only really offer my hope that you will get it sorted . goodluck
paul
although the pup numbers are normal the ratios may be relevent. you expect nn:nm:mm to be 1-2-1 but if nn:nm is 3:1 then it points to your detection method not distinguishing hets from mm homozygotes but if nn:nm is 1:2 then you are losing homozygous mutants. Even though you have a human homozygote if this change created an alternative splice site which might not be used in humans but could be active in mice perhaps?
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