Patient-derived, xenograft-expanded human BCR-ABL1+ pre–B ALL cells were cultured in vitro with or without irradiated OP9 cells as stromal support. Human full-length WT Ikaros (IK1) and the leukemia-derived DN IK6 isoform created by Rag-mediated intragenic deletion (Mullighan et al., 2008 (link)), were cloned from above patient-derived pre–B ALL cells and verified by Sanger sequencing. The two Ikaros isoforms and GFP were cloned into the TET-inducible expression vector pLVX-TRE3G-IRES (Takara Bio Inc.) with In-Fusion cloning reagents (Takara Bio Inc.). Lentiviral supernatants for expression of pLVX-EF1a-Tet3G (Takara Bio Inc.) and aforementioned cloned Ikaros and GFP-control pLVX-TRE3G-IRES were produced with the packaging vector pCDNLBH and EM140 envelope vector. Expression was induced with doxycycline, a tetracycline (TET) analogue. For Cas9/CRISPR-mediated depletion experiments, scrambled or gene-targeting gRNA sequences (Table S5) were purchased from Transomic technologies (cloned into the pCLIP-grNA-hCMV-RFP vector) and transduced into parental cells that had been stably transduced and selected for DOX-inducible expression of Cas9-IRES-ZsGreen.
Plvx tre3g ires
Manufactured by Takara Bio
Sourced in United States
The PLVX-TRE3G-IRES is a lentiviral transfer vector designed for inducible gene expression. It contains the Tet-responsive element (TRE) promoter, which allows for tight regulation of gene expression in the presence of tetracycline or its derivatives. The vector also includes an internal ribosome entry site (IRES) for bicistronic expression of the gene of interest and a reporter gene.
Automatically generated - may contain errors
Lab products found in correlation
Plvx ef1a tet3g, by Takara Bio (2 mentions)
Hifi assembly, by New England Biolabs (2 mentions)
In fusion cloning reagents, by Takara Bio (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Tet on 3g inducible expression system, by Takara Bio (1 mentions)
Plvx tet3g vectors, by Takara Bio (1 mentions)
Puromicin, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Y 27632, by Takara Bio (1 mentions)
Polybrene, by Merck Group (1 mentions)
6 protocols using plvx tre3g ires
1
Ikaros Isoforms Regulate Pre-B ALL Cell Fate
2
Inducible Ik1 Isoform Expression
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Cloning and cell line generation was performed as described previously [29 (link)]. Briefly, full length Ikaros isoform (Ik1) with IRES-GFP and an empty vector IRES-GFP control were cloned into the TET-inducible expression vector pLVX-TRE3G-IRES (Takara Bio, San Jose, CA, USA) and transduced into B-ALL cells expressing TET activator pLVX-EF1a-Tet3G (Takara Bio, San Jose, CA, USA). Expression of wild-type IK1 and GFP control samples was induced with the addition of 1 ug/mL doxycycline, a TET analog.
3
Lentiviral Transduction of ERG Variants
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For transfection-based reporter assays, human GR and MR were cloned into pACT (Promega) as N-terminal VP16 fusions using HiFi assembly (NEB). The human ERG ETS domain, and human VP16-AR WT and mutants were cloned into pCDNA3.1 using HiFi assembly. All other constructs were described previously (Wasmuth et al., 2020 (link)). For lentiviral transduction of ERG variants in mouse prostate organoids, modified derivatives of pLVX-TRE3G-IRES (Takara) were engineered for constitutive expression by replacing the TRE3G element with a UBC promoter. To construct LVX-eGFP-ERG-PuroR variants, eGFP was cloned into MCS I; human ERG variants were cloned via HiFi assembly into MCS II.
4
Lentiviral Transduction of ERG Variants
5
Inducible Expression of Cdc42 in Glioma Cells
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The Tet-On® 3G Inducible Expression System containing pLVX-TRE3G-IRES and pLVX-Tet3G vectors was purchased from Clontech (Mountain view, CA). The Cdc42 construct (pCMVXL5-Cdc42) was generated by SIDNET at The Hospital for Sick Children. Briefly, the Cdc42 open reading frame was cloned into the pLVX-Tet3G vector via BamHI and NotI sites and packaged into lentiviruses [50 (link)]. Cells infected with viruses were selected in medium containing puromycin at 0.4 μg/ml for U87MG and U251MG. Doxycycline (Sigma-Aldrich) at 500ng/ml was used to induce Cdc42 expression in cell culture. Cdc42 activation assay and western blot analysis were performed to verify the construct expression level.
6
Establishment of Inducible Gene Expression in hESCs
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The ORF sequence of ENSG00000205704 and the human codon-optimized HA × 3 sequence were cloned into pLVX-TRE3G-IRES (Clontech, 631362), which was subjected to the production of the lentivirus stock. For transduction, concentrated viruses were added into the hPSC medium (MOI 10) and incubated with polybrene (Sigma, 8 μg ml−1) and Y-27632 (10 ng ml−1) for 24 h. The virus packaged with pLVX-EF1a-Tet3G constructs (Clontech, 631359) were also co-tranducted into hESCs to built the Tet-on system. After selection with G418 (Sigma, 400 μg ml−1) and puromicin (Sigma, 0.5 μg ml−1) for 2 weeks, individual colonies were picked and further genotyped with real-time PCR assays under 2 days of doxorubicin (Sigma, 1 μg ml−1) treatment (primers: ORF-RT-F and ORF-RT-R).
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