We created parent mice with long trinucleotide repeats. We did genotyping on offspring mice and it worked well. But the most recent generation showed no band at all. We checked the genotype of parents mice and nothing's wrong. Why this generation were all negative? Could that be related with the age of parent mice? All protocals used were unchanged.
If the parents are heterogeneous then some offspring can potentially be negative.
And if you are dealing with small number of samples then it is not unusual to get all of them negative. Only remedy is to look for other offsprings and see if they turn out to be right.
Or it could be an issue of your genotyping method.
May be the water bath is not working that deactivates the proteinase K after lysis of tissues.
Thank you for replying. There were about ten offspring and all of them were negative, so I think it’s not about chance. And you are right, maybe it’s genotyping issue.
By the way, do you think it is possible that there’s some sort of unstableness on long repeat sequence that lead to gene loss in offspring?
Gene instability in offsprings may be related to the genetic recombination during the generation of sperms and OVA. However, it is also a random event which should not cause a uniform deletion of a particular gene in all the offsprings.
You should investigate your genotyping further.
Most probably, the inactivation of proteinase-K in lysis buffer, post digestion of tissues.
Complete inactivation of proteinase-K occurs at temepratures over 85 degree centigrade with incubation over 2 hours.
If for any reason the proteinase-K remains active, it will cause degradation of the DNA polymerase used for PCR for genetyping.
Hi Chenyu,
Just one comment on genotyping.
It is always good to include controls, both positive and negative, when genotyping. In your case, for each PCR reaction there should be a positive control, which is a gDNA sample that you know for sure will work (e.g. gDNA from the parents or previous offspring), and a negative control that gives your nothing, which is typically using ddH2O as template with everything else the same.
If you see the positive control works, but your testing samples do not, then another test you can try is to do a PCR using primers targeting a conserved region/gene, which you know will not be affected by this particular genetic modification. If your testing samples work in this second test, then it is likely that there is something wrong with the genotype of this batch of offspring at the modified locus. If again the controls worked but not your testing samples, then it is most likely there is something wrong with your samples, such as gDNA preps.
Hope it helps. Good luck!
Best,
Sheng