Exon 3 is deleted in this cell line. This means that quite a big chunk of p53 is still present and also quite a few splice variants of p53 are still expressed. Jean Christophe Bourdon's group has been working on this cell line and I am sure he can tell you precisely which variants are still expressed. One of the people in his group showed data with this cell line in the last p53 conference in Stockholm a few weeks ago.

We have also used this cell line and did not see any p53 expression using the the anti-p53 DO1 antibody, which recognises aa 11-25 of human p53. If you see some expression in the p53 -/- cells, you should make limiting dilutions and assess clones.

As the DO-1 only recognises the N-terminus it is not surprising it doesn't see any full length p53. Try CM1, Fl393 or Ab421 and you will see the N-terminal truncated isoforms running lower than full length.

Exon 2 encompasses aa 1-24. Thus, I assumed that if exon 3 was deleted, at least some of the p53 variants would have exon 2. Therefore, I looked at how Bunz designed the targeting vector: "The targeting vectors used in this experiment were constructed so that the first codons of the drug resistance markers replaced the first codon of the p53 gene within exon 2."

This explains why DO1 fails to detect any p53 variants in the KO cells. However, antibodies against aa 32-79 also give no signal. This region lies in exon 4, which contains ATGs that may serve as starting Met. Thus, p53 splicing variants in the -/- cells should miss exon 1-3 and have compromised DNA-binding abilities. But.... this is just in theory.

Thank you very much for your help, Patricia and Metello - it's very useful to have your input!  Like Metello, we have seen nothing with the DO1 antibody, but did see a residual band with the (C-terminal) FL393 antibody.  With our phenotypic assays the p53 seems largely inactive, but since we saw a band I wanted to check.

Thanks again!

Thank you to all for the input on this question. I was also wondering this question, as we do not detect p53 protein using DO1 antibody but can still detect gene expression using an ABI qPCR probe (~28-29 Ct in KO vs. ~25-26 Ct in WT/parental).

sorry to answer late to this question. HCT116 p53-/- are deleted of the exon2. We verified by PCR however, the HCT116 p53-/- still express all the p53 isoform (D40p53alpha,beta,gamma), d133p53alpha,beta,gamma and also the D160p53alphabeta and gamma)

The p53 protein isoforms are abundant using CM1 (polyclonal antibody with multiple epitope). the DO1 does not detect p53 proteins as it sepitope is encoded by exon2.

I published that in 2006 and in 2011 (Murray-Zmijewski et al, 2006 Cell death differentiation and Aoubala et al., 2011 Cell death Differentiation). We have papers in preparation showing the roles of the p53 isoforms in HCT116 p53-/-.

Hi jean-Christophe,

Thanks for your answer - your paper sounds really interesting, please do let me know when it comes out!

cheers,

Dan

We are trying to acquire these cells for an experiment or two, but are having trouble tracking them down. Does anyone know a good contact for HCT116 cells with and without p53? I'm happy to pay shipping or any other associated costs, obviously.