Hello,
I recently did p53 CRISPR/Cas9 Knockout on MCF10A cell line.
I would like now to do a p53 functional assay, in order to see if p53 still have or not a function on the cell. I'm thinking on inducing DNA damage (protocols?) and see the expression of p21, for example. What can you advise?
Thank you so much in advance.
Hi
Dear friend
There are different methods for p53 like qpcr, Western blot, CISH and immunohistochemistry.
I think u need 2 kind of different methods 1 for Gene expression and other for protein activity for example you can use pcr and immunohistochemistry or you can use CISH and Western blotting on the other hands you can use pcr and western.
The advantage of using 2 method is proof of Gene and protein.
Good luck
Molavi
p21 can be activated independently of p53, so your results might be inconclusive.
If you have completely KOed p53 from mice, there should be no activity. If you are not certain you can use qPCR, western blot, IHC and so forth to look at gene and protein expression
As an important function of p53 is to act as a transcriptional transactivator of a large number of target genes. It is this property that is tested by the FASAY.
Or challenge your cells with DNA damaging agents and carry out Immunoblot analysis to detect mutant and wild-type p53 protein by immunoprecipitation.
I think Northern blot is also relevant to isolate total RNA from subconfluent cultures and resolve by electrophoresis.
p21 is responsive to many factors so no reliable marker of p53 activity.
Most people check first by PCR whether the gene of interest is gone and validate by showing antibody-based proof of lack of protein (Western blot? Immunofluorescence?). But if you are to perform qPCR anyway, just compare the p53 levels of the WT versus p53 KO cells...
Dear Irina Fonseca
If you have used the CRISPR/Cas9 to KNOCKOUT the p53 gene, then you could evaluate this by following Dr. Muriel Lize's suggestions.
However, if you have used CRISPR/Cas9 to make some SPECIFIC MUTATIONS on p53 and would now like to know if this mutation affects its function (particularly during DNA damage response or repair, I assume), then please read the below.
We know that p53 protein levels get stabilised upon DNA damage. So, may be to start with, you might think of looking at p53 protein stabilisation upon DNA damage.
Further, p53 protein is known to form tetramers. There are reports suggesting that tetramerization domain is essential for various functions including protein-protein interactions, post-translational modifications, and p53 degradation.
So, you could also think of investigating if your mutated p53 protein is now able to form these tetramers and extrapolate these results to its potential functions.
Good luck..!!!
Satish
Hi Irina,
just running a western might not be very conclusive, if the cell line has low baseline levels. So inducing p53 is a good idea. I'd use nutlin-3, but you can also use actinomycin D, doxorubicin, or other chemo drugs. If these don't manage to induce/stabilize p53 as compared to control it's a good phenotypic prove of your KO.
I'd also Sanger seq p53. If you did singe cell clones, you'll get a clear sequence and you'll know because of which mutation there's not p53 expression. If you're working with the bulk however, you will most likely need to do NGS to get a better resolution.
Hope that helps,
Thomas
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