Hello there, genotype matters is not something I know much about, so I decided ask about my situation here hoping that I could get some insights.
I am working on some immune receptor and I am using 2 KO strains; one obtained by CRISPR and the other one is more traditional ES mice. Apart from that, the strain (C57), diet, age, gender, housing... Everything is the same! Interestingly, these 2 KO mice show different metabolic phenotype. One put more body weight, more insulin resistance, etc. than other counterpart.
I am really confused what might be the reason to that. Is it do you think some changes by the CRISPR off-target effects?
I am talking to a sequencing service. Maybe I will send some samples for whole-genome sequencing, but it is pretty expensive. Do you think how many mice do I need to sequence for a healthy comparison? 1 from each enough? Or is there any other way you could suggest apart from sequencing?
I appreciate any comment, thank you very much.
Hi Fahrettin,
What you are describing is relatively common. I recommend you look for your gene at http://www.mousephenotype.org/ to see if other knockouts have been subjected to systematic phenotyping by the IMPC.
If what you say is correct (both are pure C57Bl/6 background), there can be many explanations including CRISPR off-target effects. Try looking at flanking genes to see if it would make sense that introducing genetic elements might have disturbed transcription factor binding sites, then check the expression of those genes by qPCR. It's good also to look carefully at the targeting strategies to be sure that they will be complete knockouts and that all mRNA variants will be affected including those with alternative start codons.
ES cell model: Gene targetings only mutate "one" of the two alleles. Meaning: you still have WT allele.
CRISPR: Mutate one or both alleles plus most probably you will get mosaicism.
If you wonder about off-targets, check out the possible off-target sites and design primers flanking those regions, include MOCK and WT to your reaction.
I see no reason to do WGS. I believe the difference comes with the WT allele.
Cheers ^^
Are you sure the same DNA sequence of the target gene is deleted in your two mice? If not, the mice may not show the same phenotype because two different isoforms are activated.
Hi Fahrettin,
What you are describing is relatively common. I recommend you look for your gene at http://www.mousephenotype.org/ to see if other knockouts have been subjected to systematic phenotyping by the IMPC.
If what you say is correct (both are pure C57Bl/6 background), there can be many explanations including CRISPR off-target effects. Try looking at flanking genes to see if it would make sense that introducing genetic elements might have disturbed transcription factor binding sites, then check the expression of those genes by qPCR. It's good also to look carefully at the targeting strategies to be sure that they will be complete knockouts and that all mRNA variants will be affected including those with alternative start codons.