Validation of polyclonal KO cell line without antibody

Really hope someone can help! I recently made a few different knock out cell lines using pLentiCrisprv2 protocol from the Zhang lab. 2 of these have been successful and were validated using good antibodies.

1 of the other genes I was knocking out has many different splice isoforms and the antibodies to this protein are terrible so I cannot tell if there is a knock out.

I need to know what the best method of validating this KO cell line would be, considering it is a polyclonal cell line. From my understanding, Sanger sequencing will not work as there will be too many different sequences.

I have tried qPCR and one of the isoforms appears to be gone but another isoform seems to be still present. What should I be looking out for when doing a qPCR to validate these lines?

Considering the indel will only be a few bp, will qPCR be able to detect changes at all?

Any other method that could help me validate this cell line?

Thanks so much


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Lemon Mummyover 2 years ago

Our lab uses 'ICE TIDE' which works reasonably well. It is based on sanger sequencing - but it deconvolutes all of the multiple sequences. It is reliable for detecting the approximate number of KOs in a polyclonal pool of edited cells.

https://www.synthego.com/products/bioinformatics/crispr-analysis

I would be interested to hear if anyone else uses something else more reliable though.

Cyan Wendigoover 2 years ago

Are you able to give me a protocol of how you prepare your samples for Sanger sequencing, do you sequence the PCR product directly or do you insert into another vector prior to sequencing? Is ICE TIDE a web application where you input your sequencing data or are there other steps involved?

I'd also be interested in hearing any other methods people use when antibody is not available for a polyclonal KO population?

Lemon Mummyover 2 years ago

What we do is amplify approximately 300-800bp around the target cut site, then if you get a nice clean band clean it up and sequence that directly. You must also sequence a WT sample for the reference. Then upload both the WT and KO samples, and it will give you the result. It's a web app, its very simple and I think quite self explanatory when you get there.

Cyan Wendigoabout 2 years ago

I had one more question. Was your WT PCR very messy? My WT sequencing results are so messy I cant compare anything with it! I was in a rush to get the results and so the PCR product was purified with a PCR cleanup kit but I dont' know if thats enough? is gel purification absolutely necessary?

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