Retroviral and Lentiviral Construct Generation

Retroviral pBabe-puro-based human wt/mutp53 overexpression and lentiviral pLVHMshp53 vectors were described previously^{81 (link),82 (link)}. wt/mutp53s were synthesized by polymerase chain reaction (PCR) and cloned into an XbaI-digested Flag-HA-pcDNA3.1 vector using the In-Fusion cloning strategy (Takara Bio) to get Flag and HA double-tagged constructs. Similarly, MCMs were synthesized by reverse transcription PCR and cloned into EcoRI-digested pcDNA3.1/V5-His-A or pLVX-IRES-mCherry vectors using In-Fusion cloning. A TP53 CRISPR/Cas9 knockout plasmid was obtained from Santa Cruz Biotech. Murine R270H mutp53 was generated by site-directed mutagenesis using pMXs-p53 as a template and then cloned into a pLVX-IRES-hygro vector using In-Fusion cloning. The sequences of all the constructs were validated by sequencing analyses. Lentiviral short hairpin RNAs (shRNAs) against MCM5, CGAS, STING1 (TMEM173), and human and murine RelB were purchased from Dharmacon or Sigma-Aldrich. Information on all the vectors, primers, and shRNAs is listed in Supplementary Data 3.

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Zhao M., Wang T., Gleber-Netto F.O., Chen Z., McGrail D.J., Gomez J.A., Ju W., Gadhikar M.A., Ma W., Shen L., Wang Q., Tang X., Pathak S., Raso M.G., Burks J.K., Lin S.Y., Wang J., Multani A.S., Pickering C.R., Chen J., Myers J.N, & Zhou G. (2024). Mutant p53 gains oncogenic functions through a chromosomal instability-induced cytosolic DNA response. Nature Communications, 15, 180.

Publication 2024

In-Fusion cloning strategy

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Overexpression of wild-type (wt) or mutant (mut) p53
Knockdown of MCM5, cGAS, STING1, and RelB using shRNAs

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: Flag and HA double-tagged constructs for wt/mutp53 overexpression
Negative control: TP53 CRISPR/Cas9 knockout plasmid

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