Plasmid of pLenti CMV TetR Blast (716-1) (#17492, Addgene, USA) was purchased from Addgene. pUC57 plasmid containing NLRP3-CASP1 gene (PUC57-CASP1-T2A-NLRP3-R260W), pUC57 plasmid contains ASC gene (pUC57-MYC-ASC) and CASPorter plasmid was synthesized and purchased from GenScript. CD514B-1/NLRP3-CASP1 construct was generated by inserting CASP1-T2A-NLRP3-R260W cDNA from PUC57-CASP1-T2A-NLRP3-R260W plasmid as a Nhel-BamHI fragment into the lentiviral backbone pCDH-CMV-MCS-EF1α-Neo/CD514B-1 (empty vector; System Biosciences, USA). pLenti-puro/myc-ASC construct was generated by inserting MYC-ASC cDNA from pUC57-MYC-ASC as an EcoRV-DraI fragment into the tetracycline-inducible pLenti-Puro lentiviral vector (#39481, Addgene, USA). The correctness of insertion was confirmed by digestion with EcoRV/DraI and DNA sequencing (Hartwell center, St. Jude Children’s Research Hospital, USA).
Plenti cmv tetr blast 716 1
Manufactured by Addgene
Sourced in United States
The PLenti CMV TetR Blast (716-1) is a lentiviral vector that expresses the Tetracycline Repressor (TetR) protein under the control of the CMV promoter. This vector allows for the inducible expression of genes of interest in target cells.
Automatically generated - may contain errors
2 protocols using plenti cmv tetr blast 716 1
1
Generating Lentiviral Constructs for NLRP3-CASP1 and ASC
2
Generating Tetracycline-Inducible Cell Lines
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cMYC and PIK3CAH1047R (cloned from Addgene plasmid #12524) alleles were first cloned into pENTR4 no ccDB (686-1) (Addgene plasmid #17424) and subsequently shuttled into pLenti CMV/TO Puro DEST (670-1) (Addgene plasmid #17293) and pLenti CMV Hygro DEST (w117-1) (Addgene plasmid #17454), respectively (Campeau et al., 2009 (link)). Tetracycline-inducible cells were generated using pLenti CMV TetR Blast (716-1) (Addgene plasmid #17492). Lentiviral particles were generated using HEK293Tv (kind gift from Sam Benchimol) and MCF10A cell lines were transduced at the same time and with an equivalent viral titre.
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