Purpose is to study the impact of mutated p53 protein in tumorigenesis.
hTERT alone isn't sufficient to immortalize human epithelial cells while other methods known to my knowledge turns down p53. Is there any ways for immortalization keeping p53 safe?
Precise answers would be highly appreciated.
Thank you.
If the goal is to retain the "functional activity" of p53 in human epithelial cells at normal temperature, I would consider introducing hTERT and and a temperature-sensitive mutant of p53. At non-permissive temperature (39 degree C), introduced p53 would be mutant and would immortalize cells. At permissive temperature (32-37 degree C), p53 would be functional. This cell model system (see link for details) will retain the p53 functions in cells.
link: https://www.ncbi.nlm.nih.gov/pubmed/17534448
The p53 mutation I'm handling is not temperature sensitive! Do you think Rb knockdown along with the introduction of hTERT will give me immortalized epithelial cell?
I don't think that Rb knockdown wil be sufficient, and Rb is deregulated anyway when you already have the hTERT. But depending on the context of your studies you could overexpress an oncogene such as the large T oncoprotein of the SV40 virus or oncogenic Kras or Myc. Here: http://www.nature.com/onc/journal/v21/n29/full/1205550a.html. Overexpressing DNp73 would also inhibit p53's functions. But definitely the best would a temperature-sensitive mutant as proposed by Prof. Choubey.
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