I am working with some human WT and receptor KO IPSCs (mono & bi-allelic clones). The knock-outs have been engineered in a core facility by means of single bp deletions. Having expanded & differentiated these IPSCs, I now need to confirm that the KO has actually been effective c/w WT and because only a single bp deletion causing a frame shift, qPCR won't be helpful in demonstrating this.
(Sanger sequencing maybe the only option?)
I am therefore using FACS and WB to demonstrate absence or non-functional translated protein at protein level. I have a number of options of antibodies to try however I would like to use an online genetics tool/program to find out which specific epitopes each antibody binds to if this is possible? I have the information on which Exon, CRISPR sequences, base number, start codon etc but in very unfamiliar territory if anyone can help point me in the right direction and I'm a bit overwhelmed by the options/tools available!
Many thanks in advance
Hi Hatty,
The first thing to do is indeed Sanger sequencing. However, this must have been done by the facility. Otherwise, you should not know the specific genotype (single bp deletions).
It is also important to know, whether the targeted exon is essential for the function of the protein and, or whether there are alternative transcripts without this exon. Ensembl.org will give you a first idea.
In case that the exon is essential, or no transcripts without this exon exist, a simple Western with any functional verified antibody will do the job. If you are using a commercial antibody, information about the antigen should be available.
Hi Hatty,
For confirmation of KO at protein level you can simply do Flow cytometry with any specific antibody. Because, if a frame shift- mutation is intended, then it is very likely that the protein is not expressed. This you can observe by flow cytometry by presence or abscence of the signal related to the receptor.
Second method is sequencing and I believe there are several companies that do sequencing, which are not that costly at gene level.
Genewiz does it in USA.
You may simply compare the DNA sequences of WT and the KO clones.
good luck,
S
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