The YAP inducible-knockdown vector was constructed from the shYAP1 sequence (Addgene plasmid 27368) and the Tet-pLKO-puro backbone (Addgene plasmid 21915). The 4xGTIIC-Nluc Hippo Reporter vector was constructed by direct PCR from a synthetic TEAD luciferase reporter (Addgene plasmid 34615) [24 (link), 25 (link)], NanoLuc® genetic reporter vector (pNL1.1[Nluc]; Promega), and pSicoR PGK puro backbone (Addgene plasmid 11586). The helper plasmids psPAX2 and pMD2.G (Addgene plasmids 12260 and 12259) were contransfected with the vectors into HEK-293TN cells (System Biosciences) for lentivirus particle assembly. Media containing virus were collected at 48, 72, 96 and 120 hr after transfection. The supernatant was first condensed using Lenti-X™ Concentrator (Clontech) according to the manufacturer’s instructions for storage at −80°C and then used for hPSC infection in the presence of 6 µg/mL polybrene (Sigma). Transduced cells were cultured on Matrigel in mTeSR1 for at least 3 days and then selected and clonally isolated based on resistance to at least 1 µg/mL puromycin in mTeSR1.
Tet plko puro backbone
Manufactured by Addgene
Sourced in United States
The Tet-pLKO-puro backbone is a lentiviral vector that allows for inducible gene expression in mammalian cells. It contains a puromycin resistance cassette for selection of transduced cells and a tetracycline-responsive promoter for regulated gene expression.
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Lab products found in correlation
Polybrene, by Merck Group (2 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Tead luciferase reporter, by Addgene (1 mentions)
Hek293tn cells, by System Biosciences (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Stbl3 e coli, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Shrna1, by Merck Group (1 mentions)
Shrna2, by Merck Group (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Plenti cmvtight puro dest, by Addgene (1 mentions)
5 protocols using tet plko puro backbone
1
Lentiviral Vector Construction and Transduction
2
Engineered Inducible Protein Dimerization
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pHR-SNAP-CD86-mOrange-FKBP plasmid was a kind gift from Ricardo A. Fernandes. Tet-pLKO-puro backbone for tetracycline-inducible was obtained from Addgene (accession number #21915). For live cell imaging, pHR-SNAP-CD86-eGFP-FKBP, pHR-eGFP-CD86-HaloTag-FKBP and Tet-pLKO-puro SNAP-CD86-eGFP-FKBP(f36v) were created using Gibson assembly. For AP20187-induced homodimerization of FKBP, we introduced a point-mutation (FKBPf36v) following previously published protocols14 ,35 (link) and created pET30-10His-FKBP(f36v)-SNAP using Gibson assembly. Plasmids were deposited at Addgene (accession numbers #200280-#200283).
3
Inducible shRNA Knockdown in DLD-1 Cells
4
Conditional PD-L1 Knockdown in CT26 Cells
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Five PD-L1-specific shRNA (shRNA1, shRNA2, shRNA3, shRNA4, shRNA5) and a scrambled control (Table 1 ) from Sigma (St Louis, USA) was cloned into a Tet-pLKO-puro backbone purchased from Addgene (Cambridge, UK) respectively. The plasmids were mixed with XhoI (Thermo Fisher Scientific, Massachusetts, USA) at 37°C for 15 min. Cleaved DNA fragments were separated on 2% agarose gels to identify successful cloning. These verified plasmids were transfected into packaging cells and then virus-containing medium supernatants were used to infect CT26.WT tumor cells using polybrene methodology. For conditional knockdown, stable cell lines were generated after screening with 8 μg/mL puro for three weeks. The expanded surviving cells were treated with 200 ng/mL Dox and 100 ng/mL IFN-γ for 48 h. The efficiency of PD-L1 knockdown was detected by western blot.
5
Dox-Inducible Plexin-B2 Overexpression and Knockdown
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A plasmid for a lentiviral vector for doxycycline (Dox)-inducible human Plexin-B2 overexpression was generated by inserting human PLXNB2 cDNA by Gateway cloning into pLenti-CMVtight-Puro DEST (Addgene #26430). Target cells were coinfected with a lentivirus expressing Tet-On 3 G transactivator protein and hygromycin resistance (VectorBuilder, VB160122 -10094). Stable PLXNB2 Tet-On hESC lines were established by puromycin (1 μg/ml) and hygromycin (200 μg/ml) selection of coinfected cells.
Temporally controlled KD of Plexin-B2 at different phases of hESC colony expansion was achieved with TET-ON lentiviral vectors (Tet-pLKO-Puro backbone, Addgene #21915, deposited by Dimitri Wiederschain) expressing Dox-inducible shRNA targeting PLXNB2 and puromycin resistance marker (pLKO-Tet-On-PLXNB2–shRNA1 and –shRNA2). A non-targeting shRNA vector (pLKO-Tet-On-shRNA-Control) served as control (oligonucleotides used for construction of shRNA vectors are listed in file Supplementary Data1 ). Stable PLXNB2 shRNA lines were established by puromycin selection (1 μg/ml) over 7 days. The PLXNB2 overexpression and the shRNA-mediated PLXNB2 KD were induced by adding 1 μg/ml Dox (MP Biomedicals) to the culture medium.
Temporally controlled KD of Plexin-B2 at different phases of hESC colony expansion was achieved with TET-ON lentiviral vectors (Tet-pLKO-Puro backbone, Addgene #21915, deposited by Dimitri Wiederschain) expressing Dox-inducible shRNA targeting PLXNB2 and puromycin resistance marker (pLKO-Tet-On-PLXNB2–shRNA1 and –shRNA2). A non-targeting shRNA vector (pLKO-Tet-On-shRNA-Control) served as control (oligonucleotides used for construction of shRNA vectors are listed in file Supplementary Data
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