Our lab just obtained some 5XFAD Het mice that we plan to use. Our plan is to use WT, Hets, and Homozygous animals and was wondering if anyone had a better way to genotype them besides using real-time PCR (which is really expensive). Anyone has any primers suggestions that we can use for normal PCR?
You could generate a number of primer sets where the heterozgous base or the deleted bps of the vaious alleles are placed at the 3' end of one of the primers (perhaps even generate primers for each variation to be thorough). When a primer binds to a sequence where the 3' end is mismatched, as long as the annealing temperature is high enough you shouldn't get (or should significantly reduce) any extention from that primer hence no PCR product. In this way you can then discriminate between various alleles but to do so confidently you really need to spend time optimising the conditions and as such it would help if you had clones of the various sequences. If you don't have them you could generate from the heterzygous mouse. TIme is money though so I guess you can take your pick!!
HI,
YOu can design Allele Specific PCR, Cleaved Amplified Polymorphic Sequences (CAPS) - a PCR-RFLP. You can find Restriction Enzymes using online tools available like NEB cutter, web cutter etc.
Our lab uses regular PCR primer genotyping on tail samples (standard Jackson Labs suggested primers). But to be honest, they don't appear to be completely reliable as we're currently reevaluating their results.
It's laborious and expensive but we're also using a pan-A-beta antibody via immunohistochemistry on some frozen brain slices to assess pathology, as well as qPCR using hAPP primers. Those approaches work much better and allow you to verify that pathology is present at whichever time point you select. Not the most cost effective but certainly more reliable for us at the moment.
Edit: After some tweaking of temperatures and primer concentrations (we had to use the internal control primers at 2.5x concentration of the 5XFAD positive band primer set), we've got the Jax primers working reliably. We also genotype at weaning and post sac to make 100% sure of genotype.
Homozygous vs hemizygous. Much of the research was completed with only one copy (hemizygotes) of the mutant genes. Since an extra copy has additive phenotype and pathologic effects you may need to avoid homozygotes if you cannot afford RT-PCR for genotyping. Sorry, its not a big help but I could not think of any solution without breeding two independent 5xFADs that have distinct differences near the mutant gens on Chromosome 3. That would only be MORe expensive. Luckily, a breeding strategy will restrict homozygotes very well. Can you do your studies with Hemizygotes alone?
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