The B16-F10 luciferase cell line was generated by stably transfected with luciferase expressing vector pSBbi-pur (Addgene #60523) and pCMV (CAT)T7-SB100 (Addgene #34879) by Lipofectamine 2000 (Thermo Fisher). C57BL/6 (8–12 weeks old) male mice were inoculated with 5 × 105 B16-F10 cells. Twelve days later, mice were injected with engineered macrophages. Four days after macrophage injection, XenoLight D-Luciferin Potassium Salt (PerkinElmer) was intra-peritoneally injected at 10 μL/g of body weight for each mouse. Images were detected by an IVIS Lumina II in vivo imaging system (PerkinElmer, Thermo Fisher, US) 10–15 min after injection.
Pcmv cat t7 sb100
Manufactured by Addgene
Sourced in United States
PCMV(CAT)T7-SB100 is a plasmid vector that contains the constitutive cytomegalovirus (CMV) promoter, a T7 promoter, and the SB100X transposase gene. This plasmid is commonly used for the expression of genes of interest in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Puromycin, by Thermo Fisher Scientific (6 mentions)
Psbbi rp, by Addgene (3 mentions)
Psbbi pur, by Addgene (2 mentions)
Psbtet gp, by Addgene (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Psbbi rb, by Addgene (2 mentions)
Psbtet pur vector, by Addgene (2 mentions)
Psbbi gn, by Addgene (2 mentions)
Puromycin, by Cell Signaling Technology (2 mentions)
Anti sox2 d6d9, by Cell Signaling Technology (2 mentions)
Tracrrna atto 550, by Integrated DNA Technologies (2 mentions)
Custom crrnas, by Integrated DNA Technologies (2 mentions)
Silentfect lipid reagent for rnai, by Bio-Rad (2 mentions)
Ivis lumina 2 in vivo imaging system, by PerkinElmer (1 mentions)
Xenolight d luciferin potassium salt, by PerkinElmer (1 mentions)
Pei max, by Polysciences (1 mentions)
Pmc cmv, by System Biosciences (1 mentions)
Pt caggs nrasv12, by Addgene (1 mentions)
Effectene, by Qiagen (1 mentions)
27 protocols using pcmv cat t7 sb100
1
Bioluminescent Melanoma Tumor Imaging
2
Tetracycline-Inducible TCF4 Expression
3
Engineered Protein Localization Constructs
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MicroID2 and TurboID constructs were cloned into the sleeping beauty backbone pSBbi-RB (Addgene; plasmid no.: 60522). Site-directed mutagenesis or restriction enzyme cloning was utilized to introduce localization sites: 3× nuclear localization sequence (NLS) (Addgene; plasmid no.: 98875), mitochondrial targeting sequence (MTS) (Addgene; plasmid no.: 98876), and the nuclear export sequence (NES) (Addgene; plasmid no.: 49386). The final constructs were transfected into BEAS-2B and cotransfected with the sleeping beauty transposase (pCMV(CAT)T7-SB100; Addgene; plasmid no.: 34879) at a 1:5 ratio. At 4 to 5 days post-transfection, cells were selected by puromycin A1 treatment (2 μg/ml). After selection, integration was confirmed by measuring FLAG expression and biotinylation via immunofluorescence and Western blotting.
4
Cloning Human Furin in Sleeping Beauty
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Human furin was cloned in the sleeping beauty transposon plasmid (30 (link)) pSB-bi-RP (Addgene, catalog no. 60513), transfected along with transposase and pCMV(CAT)T7-SB100 (Addgene, catalog no. 34879) into Vero cell using PEI Max (MW 40,000; Polysciences), and selected with 2.5 μg/mL Puromycin (Gibco). Clonal cell lines were generated through limited dilution of the polyclonal cell line on a 96-well plate at the concentration of 0.3 cell/well.
5
Minicircle Transposon for GFP and p53
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To generate a minicircle transposon encoding GFP, the synthetic SB transposase restriction sites IR/DR(L) and IR/DR(R) were ligated to the corresponding restriction sites SmaI—ClaI and StuI—EcoRV of the parental minicircle vector pMC.CMV-GFP (System Biosciences, Palo Alto, CA, USA).
A synthetic codon-optimized cDNA encoding the full 393 amino acids of p53 (Eurofins MWG Biotech, Ebersberg, Germany) fused to a T2A endoproteolytic cleavage site and a puromycin resistance gene was ligated to the corresponding restriction sites ClaI and HindIII of the parental minicircle vector pMC.CMV (System Biosciences), resulting in pMC-p53-puroR. In pMC-p53-puroR, the p53/puroR transgene was flanked by transposase restriction sites IR/DR(L) and IR/DR(R). pMC-puroR lacking the p53 coding sequence was used as mock control. The pCMV(CAT)T7-SB100 (Addgene, Watertown, MA, USA, plasmid # 34879) encoding hyperactive SB100X Sleeping Beauty transposase has been described previously [28 (link)]. The SB100X gene sequence was amplified by PCR adding XbaI restriction sites and ligated to the XbaI restriction sites in the MCS of the parental minicircle vector pMC.CMV-MCS (System Biosciences). All vector inserts were confirmed by sequencing (Microsynth Seqlab, Göttingen, Germany).
A synthetic codon-optimized cDNA encoding the full 393 amino acids of p53 (Eurofins MWG Biotech, Ebersberg, Germany) fused to a T2A endoproteolytic cleavage site and a puromycin resistance gene was ligated to the corresponding restriction sites ClaI and HindIII of the parental minicircle vector pMC.CMV (System Biosciences), resulting in pMC-p53-puroR. In pMC-p53-puroR, the p53/puroR transgene was flanked by transposase restriction sites IR/DR(L) and IR/DR(R). pMC-puroR lacking the p53 coding sequence was used as mock control. The pCMV(CAT)T7-SB100 (Addgene, Watertown, MA, USA, plasmid # 34879) encoding hyperactive SB100X Sleeping Beauty transposase has been described previously [28 (link)]. The SB100X gene sequence was amplified by PCR adding XbaI restriction sites and ligated to the XbaI restriction sites in the MCS of the parental minicircle vector pMC.CMV-MCS (System Biosciences). All vector inserts were confirmed by sequencing (Microsynth Seqlab, Göttingen, Germany).
6
In Vivo Tumor Growth Modeling
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Male C57BL/6j mice at 4–5 weeks old were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and kept in the laboratory animal center (LAC) of NHRI. The mice received 2 μg of pCMV(CAT)T7-SB100 (Addgene #34879), 10 μg of pT/Caggs-NRASV12 (Addgene #20205), and 10 μg of pKT2/CLP-AKT-LUC plasmids through HDI and were monitored for tumor growth weekly using IVIS. Detailed procedures were described in our previous study (Liu et al., 2018 (link)).
7
Doxycycline-Inducible SLC26 Protein Expression
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Plasmids encoding chicken (cPres) and zebrafish prestin (zPres) were kindly provided by Dominik Oliver (Philipps University, Marburg), and a plasmid containing human SLC26A9 (hA9) complementary DNA was a gift from Dr. Tomohiro Shima (University of Tokyo, Tokyo). These constructs were cloned into a pSBtet-pur vector (Addgene) using SfiI sites with a C-terminal mTurquoise (mTq2) tag. C-terminally mTq2-tagged human SLC26A4 (hA4) cloned in a pSBtet-pur vector was previously generated in our laboratory (40 (link)). Mutagenic primers were used to introduce missense changes in these constructs (p.S402D/E for cPres; p.S399D/E for zPres; p.A406D/E for hA4; and A390D/E for hA9). Stable cell lines that express these SLC26 constructs in a doxycycline-dependent manner were established in HEK293T cells as previously described (40 (link)). Briefly, pSB plasmids were introduced together with pCMV(CAT)T7-SB100 (Addgene) using Effectene (Qiagen), and the transfected cells were selected in DMEM supplemented with 10% FBS and 1 µg/mL puromycin (Fisher Scientific).The hA4 and hA9 constructs were also introduced to the HEK293T-mVenusH148Q/I152L cell line that was generated in a previous study (40 (link)).
8
Comprehensive Plasmid Collection for Genetic Manipulation
9
Stable Overexpression of MLKL and RIPK3
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For stable MLKL or RIPK3 overexpression constructs for human MLKL, RIPK3 and DOX-inducible RIPK3 were cloned into pSBbi-Blasticidin (Addgene plasmid #60526) and pSBtet-Puromycin (Addgene plasmid #60507) [28] (link). Cells were then transfected with 1800 ng pSBbi-RIPK3-blast or pSBbi-MLKL-blast and 200 ng pCMV(CAT)T7-SB100 (Addgene plasmid #34879) using the Neon Transfection System (Invitrogen) following the manufacturer's protocol. For stable co-overexpression of both MLKL and RIPK3, 1350 ng of pSBbi-MLKL-blast and 1350 ng of pSBtet-RIPK3-puro were used in combination with 300 ng of pCMV(CAT)T7-SB100. As a control, cells were transfected with 2700 ng of pSBbi-blast and 300 ng of pCMV(CAT)T7-SB100 for 72 h. Transfection was followed by at least two weeks of selection with puromycin (10 µg/mL) and/or blasticidin (20 µg/mL).
10
Stable Cell Lines with Cygb Overexpression
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To establish stable cell lines overexpressing Cygb, the sleeping beauty transposon system was used.30 , 31 , 32 The pT2/Cygb or pT2‐MCS‐SV Neo and pCMV(CAT)T7‐SB100 plasmids (34879; Addgene) were cotransfected into LM8‐L cells using an electroporator (Nepa Gene, Chiba, Japan) according to the manufacturer's instructions. After 48 hours, the culture medium was changed to selection medium containing 1 μg/mL G418 (Roche Life Sciences). The cells were further cultured in selection medium for 14 days, and single colonies were isolated to establish CYGB‐overexpressing LM8‐L (CYGB‐OE) cells.
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