For the generation of p53 knock-out (p53KO) cell line, CRISPR/Cas9 plasmid (sc-416469) and p53 Homologous direct Recombinant-HDR plasmids (sc-416469-HDR) were transfected into MCF10A-Plk4 cells using the Neon® Transfection system. After transfection recovery, cells were selected with puromycin in order to select p53KO stable cell line. To have monoclonal stable p53KO clones, puromycin-resistant cells were sorted into single-cell clones by using a BD FACS Aria IIu.
To validate if p53 was successfully knocked-out, cells were exposed to three different conditions to determine the functionality of the p53 protein: treatment with 1 and 3 µM of Doxorubicin (mild and severe DNA damage, respectively) for 4 h and kept for 24 h in drug-free medium and, a third condition where 1 µg/ml of Doxycycline was added to cells for 24 h (inducing extra centrosomes). After the 24 h, cells were harvested for western blot analysis. Total protein levels of p53 and p21, a downstream target of p53, was assessed by Western Blot.
To validate if p53 was successfully knocked-out, cells were exposed to three different conditions to determine the functionality of the p53 protein: treatment with 1 and 3 µM of Doxorubicin (mild and severe DNA damage, respectively) for 4 h and kept for 24 h in drug-free medium and, a third condition where 1 µg/ml of Doxycycline was added to cells for 24 h (inducing extra centrosomes). After the 24 h, cells were harvested for western blot analysis. Total protein levels of p53 and p21, a downstream target of p53, was assessed by Western Blot.
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