Lentiviruses were generated in HEK293T cells (23 (link)). Lentivirus expressing H2B-mCherry has been described (23 (link)). The virus expressing the fusion EGFP-Centrin2 was constructed from pEGFP-Centrin 2 (provided by E. Nigg; Addgene plasmid #41147) and pWPXLd (23 (link)). Lentivirus shp53 pLKO.1 puro (provided by R. Weinberg; Addgene plasmid #19119) was used to knock down p53 with pBabe-puro (provided by H. Land, J. Morgenstern, and R. Weinberg; Addgene plasmid #1764) as empty vector control.
Shp53 plko 1 puro
Manufactured by Addgene
Sourced in United States
Shp53 pLKO.1 puro is a plasmid vector that contains the shRNA sequence targeting the p53 gene. It is commonly used for gene knockdown experiments in cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Pbabe puro, by Addgene (1 mentions)
Pcmv neo bam p53 r175h, by Addgene (1 mentions)
Pcmv neo bam p53 r273h, by Addgene (1 mentions)
Mission shrna, by Merck Group (1 mentions)
Pbabe hygro htert, by Addgene (1 mentions)
Plenti cmv gfp blast, by Addgene (1 mentions)
Plpcx mito grx1 rogfp2, by Addgene (1 mentions)
Vsvg envelope plasmid, by Addgene (1 mentions)
Pmscv mlin28a, by Addgene (1 mentions)
5 protocols using shp53 plko 1 puro
1
Lentiviral Expression of Fluorescent Markers
2
Plasmid Constructs for MDM2/MDM4 Expression
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HA-FLAG-MDM4, HA-MDM2, and HA-MDM2B plasmids for insect cell and mammalian expression were described previously (24 (link), 28 (link)). His-ubiquitin plasmid (pMT107) was a gift from Dr. Dirk P. Bohmann (University of Rochester Medical Center, Rochester, NY, USA). MDM2-MDM4 RING heterodimer constructs, pETDuet-MDM2R, and pETDuet-MDM4R were generated by PCR cloning of the RING domain of human MDM2 or MDM4 into pETDuet-1 (Novagen, Madison, WI, USA). Plasmid shp53 pLKO.1 puro was from Addgene (31 (link)). Detailed information on antibodies for Western blotting (WB) analysis as well as primer sequences and conditions for RT-PCR analysis of gene expression can be found in the Supplementary Methods
3
Tetracycline-Inducible Lentiviral TRF2 Construct
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A lentiviral tetracycline-inducible TRF2ΔBΔM construct was generated by cloning the inducible TRF2ΔBΔM cassette from a pBluescript.KS vector (courtesy of Dr Lenhard Rudolph) into a neomycin resistant promoter-less lentivector (Amsbio) using X-baI restriction sites. The Tet-regulated transcriptional transactivator protein rtTA3 containing hygromycin resistance was a kind gift from Dr Iain Fraser. The lentiviral construct for p53 short hairpin RNA (shp53 pLKO.1 puro) was from Dr Bob Weinberg (Addgene plasmid #19119), and the lentiviral plasmid for SV40LT (pRRLsin-SV40 T antigen-IRES-mCherry) was from Dr Snorri Thorgeirsson (Addgene plasmid #58993). The hTERT lentivirus was supplied by Viral Vector Facility, CNIC, Spain.
4
Lentiviral Plasmids for Investigating p53 and Autophagy
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pLKO.1 lentiviral plasmids containing shRNAs against ATG5 (TRCN000000150645) and ATG7 (TRCN0000007584) were obtained from Sigma‐Aldrich (Mission shRNA). shp53 pLKO.1 puro (Addgene plasmid 19119), pCMV‐Neo‐Bam p53‐R273H (Addgene plasmid 16439) and pCMV‐Neo‐Bam p53‐R175H (Addgene plasmid 16436) were purchased from Addgene (Cambridge, MA, USA).
5
Lentiviral Transduction of Cell Lines
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The following plasmids were used to produce viruses for the various transgenic cell lines: lentiviral plasmid (Addgene no. 19119), dR8.2 packaging plasmid (Addgene no. 8455), VSV‐G envelope plasmid (Addgene no. 8454), pMSCV‐mLin28A (Addgene no. 26357), pBABE‐hygro‐hTERT (Addgene no. 1773), shp53 pLKO.1 puro (Addgene no. 19119), pLenti CMV GFP Blast (659‐1; Addgene no. 17445), pLPCX mito Grx1‐roGFP2 (Addgene no. 64977), pLenti X2 Blast/shp16 (w112‐1; Addgene no. 22261), pLKO‐RB1‐shRNA19 (Addgene no. 25640), hypoxia‐response element‐luciferase (Addgene no. 26731), and pLKO.1‐hPGK‐Neo (SHCLND‐NM_058197, TRCN0000265840, Sigma‐Aldrich). Viral supernatants were collected within the 48‐h to 96‐h window and filtered with a 0.45 μm filter (Sartorius). Virally transduced cells of the relevant types were selected with the relevant antibiotics for 3 days, and the transduction efficiency was typically about 80%.
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