Plasmids used in this study were generated using standard polymerase chain reaction (PCR) techniques and/or type II and IIs restriction enzyme cloning. Restriction enzymes, Phusion DNA polymerase, T4 DNA Ligase, and Antarctic phosphatase were purchased from NEB. WT-ACE2 and Mut-ACE2 gene fragments were codon optimized and synthesized by Thermo Fisher and cloned into a pGIPZ backbone (Open Biosciences). The Spike protein from pcDNA3.1-SARS2-Spike was a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032 ; RRID:Addgene_145032);34 (link) this gene was cloned into a modified pcDNA 3.1 backbone (Clontech-Takara) with a beta-globin intron in the 5’ untranslated region for pseudotyping lentivirus. The Tet3G transactivator (pLVX-EF1a-TET3G) and cognate TRE3GV promoter (pLVX-TRE3G) (Takara) were cloned into modified pGIPZ and pLVX (Takara) backbones, respectively. In this context, the Spike protein (Addgene plasmid # 145032) was cloned downstream of the TRE3G promoter. All plasmids used in this study were sequence-verified, and maps will be published with the final manuscript. Chemically competent TOP10 Escherichia coli were used for transformation of all plasmids and subsequently grown at 37°C.
Plvx ef1a tet3g
Manufactured by Takara Bio
Sourced in United States
The PLVX-EF1a-TET3G is a lentiviral expression vector that allows for the tetracycline-inducible expression of your gene of interest. It contains the EF1α promoter and the reverse tetracycline-controlled transactivator (rtTA3) for tight regulation of transgene expression.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna 3.1 backbone, by Takara Bio (3 mentions)
Plvx tre3g, by Takara Bio (3 mentions)
Antarctic phosphatase, by New England Biolabs (3 mentions)
Phusion dna polymerase, by New England Biolabs (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Plvx tre3g ires, by Takara Bio (2 mentions)
Dsred express2, by Takara Bio (2 mentions)
Pgipz, by Takara Bio (2 mentions)
In fusion cloning reagents, by Takara Bio (1 mentions)
5 protocols using plvx ef1a tet3g
1
Plasmid Generation for SARS-CoV-2 Studies
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
Plasmid Generation and Lentivirus Pseudotyping
4
SARS-CoV-2 Spike Protein Cloning
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Plasmids used in this study were generated using standard polymerase chain reaction techniques and/or type II and IIs restriction enzyme cloning. Restriction enzymes, Phusion DNA polymerase, T4 DNA Ligase, and Antarctic phosphatase were purchased from NEB. psPAX2 and pMD2.G plasmids were gifted by William Miller from Northwestern University and DsRed‐Express2 was purchased from Clontech‐Takara. WT‐ACE2 and Mut‐ACE2 gene fragments were codon optimized and synthesized by Thermo Fisher and cloned into a pGIPZ backbone (Open Biosystems). The Spike protein from pcDNA3.1‐SARS2‐Spike was a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032 ; RRID:Addgene_145032);[45 ] this gene was cloned into a modified pcDNA 3.1 backbone (Clontech‐Takara) with a beta‐globin intron in the 5′ untranslated region for pseudotyping lentivirus. The Tet3G transactivator (pLVX‐EF1a‐TET3G) and cognate TRE3GV promoter (pLVX‐TRE3G) (Takara) were cloned into modified pGIPZ and pLVX (Takara) backbones, respectively. In this context, the Spike protein (Addgene plasmid # 145032) was cloned downstream of the TRE3G promoter. Cloned plasmids used in this study were sequence‐verified, and maps are available in Data S1 , Supporting Information. Chemically competent TOP10 Escherichia coli were used for transformation of all plasmids and subsequently grown at 37 °C.
5
Ikaros Isoforms Regulate Pre-B ALL Cell Fate
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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.
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