Mouse Ube2s, β-Catenin-K19R, and β-Catenin-K49R ORFs were inserted into vector pCAG-puro, respectively, for overexpression in mES cells. Human UBE2S ORF was inserted into vector pcDNA4.0/TO(+) for overexpression in HCT116 cells. The Ube2s shRNAs were cloned into pSuperpuro (Oligoengine, Seattle, WA, USA) between BglII and HindIII sites, and 19-bp shRNAs with a 9-bp loop were expressed by the pSuperpuro plasmids. The two target sequences for Ube2s knockdown in mES cells are 5′-GCTACTTCCTGACTAAAAT-3′ and 5′-GGAGGTCTGTTCCGTATGA-3′. The two target sequences for Ube2s knockdown in HCT116 cells are 5′-GCATCAAGGTCTTTCCCAA-3′ and 5′-GGCGGTTATAAAGAGGCAG-3′. Transfection of plasmids was performed using Lipofectamine 2000 (Invitrogen, 11668-019).
Psuper puro
Manufactured by Oligoengine
Sourced in United States
The PSuper.puro is a laboratory equipment designed for the selective expression of genes of interest. It functions as a lentiviral vector system that facilitates the stable integration and expression of target genes in a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Psinsi, by Takara Bio (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Fugene 6, by Promega (1 mentions)
Pmx plasmid, by Addgene (1 mentions)
Pgl3 promoter vector, by Promega (1 mentions)
Psuper neo, by Oligoengine (1 mentions)
Control sirna, by Qiagen (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
11 protocols using psuper puro
1
ORF Overexpression and shRNA Knockdown
2
Transient and Stable Knockdown of Esrrb and Klf4 in E14 Cells
3
Transfection and Knockdown/Overexpression Protocols
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Plasmids were transfected using X‐tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland), Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) or electroporation. DYRK2‐specific siRNA (Qiagen, Venlo, the Netherlands; Invitrogen), CDK14 siRNA (Sigma‐Aldrich, St. Louis, MO, USA) and control siRNA (Qiagen) were used. The siRNA were transfected using Lipofectamine RNAiMAX (Invitrogen). To isolate stable transfectants, MCF‐7 cells were transfected with pSuper‐puro (Oligoengine, Seattle, WA, USA) or pSuper‐puro DYRK2 shRNA in the presence of puromycin (1 μg/mL). MCF‐7 cells were transfected with pSuper‐neo (Oligoengine) or pSuper‐neo CDK14 shRNA in the presence of G418 (500 μg/mL). For stable overexpression, we used the GFP‐C1 vector. MDA‐MB‐231 cells were transfected with GFP or GFP‐DYRK2 in the presence of G418 (500 μg/mL). Gene knockdown and overexpression were assessed by real‐time RT‐PCR.
4
Retroviral Knockdown and Expression of p53 Mutants
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To generate retroviruses encoding shRNAs against HtrA2/Omi and p130Cas, the HtrA2/Omi target sequence, 5′-CACACTGAGGATTCAAACCAA-3′, and p130Cas target sequence, 5′-GCATGACATCTACCAAGTT-3′, were cloned into a pSuper puro (Oligoengine) or pSINsi (Takara Bio Inc.) vector. pBabe Ha-RasV12 with a hygromycin selection marker and pBabe p53R175H with a zeocin selection marker were used (Kawauchi et al., 2008 (link)). pBabe p53KRKKK, which contains K305A, R306A, K319A, K320A, and K321A substitutions in NLS (O’Keefe et al., 2003 (link)), was generated from pBabe p53 with a puromycin selection marker. The mature forms of mouse HtrA2/Omi cDNA were obtained by PCR from NIH3T3 cDNA pools and cloned into a pCMV-Flag vector. pCMV-Flag-mature HtrA2/Omi was then generated with either the S142D or S306A mutation, where Ser 142 or Ser 306 of HtrA2/Omi was replaced by aspartic acid or alanine, respectively. The Mito-DsRed expression vector was obtained from Takara Bio Inc.
5
Dual-Luciferase Assay for TTP Modulation
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MEFs were plated at ≈20% confluency in 22-mm-diameter tissue culture wells in 1 mL DMEM/10% FBS. Twenty-four hours later, 0.2 μg pcDNA3-myc-F-Luc-ARE, 0.05 μg pcDNA3-myc-R-Luc, 0.3 μg pSuper.puro (OligoEngine), 0.4 μg pcDNA3-myc, and 0.05 μg of Flag-tagged TTP wild-type or mutant constructs were transfected by TransIT-X2 following manufacturer's protocols (Mirus). Twenty-four hours later, 5 μg/mL of puromycin was added for another 24 h to select for transfected cells. Cells were then lysed in 250 μL of 1× Passive Lysis Buffer (Promega) for 20 min at room temperature and 10 μL of each lysate was assayed for firefly and Renilla luciferase activities using Dual-Luciferase Reporter Assay (Promega) reagents in a NOVOstar microplate reader. In the experiments shown in Figure 4 C, 0.3 µg of pcDNA3-Flag or 0.15 μg pcDNA3-Flag-4EHP and 0.15 μg pcDNA3-Flag-GYF2 were cotransfected instead of TTP constructs. P-values were calculated with two-tailed paired Student's t-test.
6
Plasmid-based shRNA and Overexpression
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Plasmids containing shRNAs used in this study were prepared by restriction cloning of annealed oligonucleotides into pSUPERpuro or pSUPERblast plasmid backbones (Oligoengine). The target sequences of the shRNAs were TRF1 (5′-GAATATTTGGTGATCCAAA-3′) cloned into pSUPERpuro and TRF2 (5′--GCGCATGACAATAAGCAGA-3′) pSuperBLAST (Porro et al. 2014 (link)). The pLPC_TRF2_ΔBΔM plasmid (a generous gift from Dr. Titia de Lange; Addgene, plasmid 18008) was used for overexpression of TRF2ΔBΔM. The pLPC-N-MYC empty plasmid (Addgene, plasmid 12540) was used as a control.
7
BEX4 Functional Analysis via Genetic Manipulations
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Full-length human BEX4 was generated by PCR. Full-length human BEX4 was also subcloned into pGEX-KG (GST-BEX4) and pTAP (TAP-BEX4) for the GST pull-down assay and tandem affinity purification (TAP), respectively. Fragments encoding BEX4 were subcloned into pEGFP-C1 (Clontech, Mountain View, CA, USA) to generate a GFP-fused BEX4 expression vector (pEGFP-BEX4). BEX4 mutant alleles were generated by site-directed mutagenesis. Comlementary DNAs of the BEX4 wild-type (WT) and five mutant versions, in which a single serine or single threonine residue was exchanged for alanine (S3A, T29A, S35A, T94A, and T107A), were subcloned into pEGFP-C1 and pGEX-KG. The pCMV-HA-PLK1 plasmid has been published previously18 (link). To specifically knock down BEX4, short hairpin (sh)RNAs were cloned into pSuper puro (Oligoengine, Seattle, WA, USA) containing the H1 promoter according to the manufacturer’s instructions. Oligonucleotides encoding a shRNA against BEX4 (5′-GGCCATACCTAATAGGCATAT-3′), human CDK1 (5′- GGGGATTCAGAAATTGATC-3′), human PLK1 (5′-GGTCCATTGGGTGTATTCATGT-3′) or the luciferase control (5′-CATACGCGGAATACTTCGA-3′) were synthesized, and the efficiencies of each shRNA were determined by immunoblotting12 (link),18 (link),19 (link). For transient transfection, the cells were electroporated using a microporator (Digital Biotechnology, Seoul, Korea).
8
Knockdown of Drfundc1 gene in zebrafish
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In this study, shRNAs which interfered with Drfundc1 were designed online (http://rnaidesigner.thermofisher.com/rnaiexpress/design.do ). Two shRNAs – shRNA1 and shRNA2 – targeted nucleotides of Drfundc1 ORF from 77 to 99 and 217 to 237 respectively. A mismatched shRNA (shRNAmis) as well as a random shRNA (shRNAran) without any target were designed as controls. Vector pSuper-puro (OligoEngine) was used to construct shRNA expression plasmids. shRNAs or pSuper-puro were microinjected into zygotes at a dosage of 200 pg. Drfundc1 mRNA was synthesized from pCS2 + -Drfundc1 in vitro using an mMESSAGE mMACHINE SP6 transcription kit (Thermo Fisher Scientific; AM1340). To repair defects caused by Drfundc1 shRNA, 400 pg of Drfundc1 mRNA was microinjected with 200 pg of the shRNA plasmid into zygotes. Embryos with/without injection of pSuper-puro, shRNAmis, and shRNAran were treated as controls.
9
SETX Knockout in HAP1 Cells
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To generate SETX KOs in the HAP1 line, cells were transiently transfected with pX330 containing a SETX-specific sgRNA sequence, along with pSUPER.puro (Oligoengine) at a 7:1 ratio. After 24 h, cells were selected with 1 μg/mL puromycin (ThermoFisher) for 2 d before the cells were harvested and seeded as single colonies. Initially, clones were selected on the basis of a negative signal for SETX by Western blotting. The selected clones were then sequence-verified.
10
Generating ALKBH1-deficient HEK293 cells using CRISPR/Cas9
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