Fusion cDNAs containing the full-length NR4A3 coding sequence and a C-terminal-HA epitope tag were cloned into a pLenti6/V5-D-topo vector (Invitrogen). The plasmids of each deletion cDNA (ΔAF12–288 aa, ΔDBD292–364 aa, and ΔLBD398–626 aa) were generated by mutagenesis techniques. The four recombinant plasmids and a control GFP gene construct were transfected into MIN6 cells, using TurboFect Transfection Reagent (Thermo Scientific). For stable transfection, cells were grown under blasticidin selection (3 µg/ml) for over 15 days, then single clones of cells were picked and amplified. Western blotting analyses were performed to test the stability of the transfected cell lines.
Plenti6 v5 d topo vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PLenti6/V5-D-TOPO vector is a plasmid designed for the expression of genes in mammalian cells. It features a lentiviral backbone and a TOPO cloning site for rapid and efficient insertion of genes of interest. The vector allows for stable integration of the gene into the host cell genome, enabling long-term expression.
Automatically generated - may contain errors
Lab products found in correlation
Blasticidin, by Thermo Fisher Scientific (2 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Virapower lentiviral expression system, by Thermo Fisher Scientific (1 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Ant pr 1, by Thermo Fisher Scientific (1 mentions)
Qpcr lentivirus titration kit, by Applied Biological Materials (1 mentions)
Lenti x packaging system, by Takara Bio (1 mentions)
7 protocols using plenti6 v5 d topo vector
1
Generating NR4A3 Fusion Constructs
2
Lentiviral Expression of Human SRA
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The human full-length SRA cDNA was cloned and inserted into the pLenti6/V5-D-TOPO vector (4950-00, Invitrogen, CA, U.S.A.). Using the ViraPower Lentiviral Expression System (Invitrogen, CA, U.S.A.) according to the manufacturer’s instructions. The plasmid was transfected in 293FT cells for packaging and the resulting lentivirus was used to infect the desired cell line. SRA stably transfected cells were selected in media containing blasticidin (Invitrogen). The sequence map of the plasmid pLenti6/V5-D-TOPO and the overexpression fusion gene SRA have been described in previous studies [15 (link)].
3
Generating MED12 Expressing Cell Lines
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MED12 CDS fragment which amplified from a pAc-C Med12His plasmid (Addgene plasmid #49240) was cloned into the pLenti6/V5-D-TOPO vector (Invitrogen) at the BamH1 and Xho1 sites. Lentivirus was produced by transfection of HEK-293FT cells with pLenti6-MED12, psPAX2 and pMD2.G plasmid at a 0.5:0.35:0.15 ratio. The viral supernatant was collected 48 h following transfection, filtered through 0.45 μm filter, and added to MED12 KO1 and MED12 KO2 cells. The stably expressing cell lines were selected with 20 μg/ml blasticidin for 5 days and named as KO1-MED12 or KO2-MED12.
4
Cloning and Expression of CsgA Protein
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E. coli genomic DNA was isolated from ∼1 mL of overnight culture using QIAamp DNA Mini Kit (QIAGEN), following the manufacturer’s protocol. C-terminally hexahistidine-tagged CsgA without N-terminal SEC secretion signal sequence (Evans et al., 2015 (link)) was amplified via standard PCR method using the following primers: csgA F: 5′-TCAAGGGAATTCACCATGGGTGTTGTTCCTCAGTACGG-3′, csgA R: 5′-TCAACGGGATCCCTAGTGATGATGGTGGTGA TGGTACTGATGAGCGGTCGCGTTG-3′. Thermal cycling program used for the PCR is as follows: 94°C, 5 min → 33x [94°C, 30 s → 58°C, 30 s → 72°C, 30 s] → 72°C, 7 min → 4°C. The resulting PCR amplicon was gel-purified using QIAquick Gel Extraction kit (QIAGEN) and then assembled into pLenti6/V5-D-TOPO vector (Invitrogen) following the manufacturer’s protocol. The cloned plasmid was subsequently transformed into One Shot TOP10 chemically competent E. coli (Invitrogen) and spread on LB + Ampicillin (100 μg/mL) agar plate for selection. Several plasmid clones were isolated using QIAprep Spin Miniprep kit (QIAGEN) and then screened via agarose gel electrophoresis after digesting them with EcoRI and BamHI. The correct clone was sequenced using CMV-F primer (5′-CGCAAATGGGCGGTAGGCGTG-3′) and named pLenti6-V5-D-TOPO:csgA-6x His.
5
Inhibition of LINC00667 in A549 Cells
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Sequences of sh-RNA targeting LINC00667 (sh-LINC00667), LINC00667 and miR-143-3p were subcloned into the pLenti6/V5-D-TOPO vector (Invitrogen). The nonsense sequences were also subcloned into the pLenti6/V5-D-TOPO vector, and formed the control sh-NC for sh- LINC00667, the control NC for LINC00667, the control Mock for miR-143-3p. Then, the constructed Lenti-sh-HOTAIR vectors were transfected into A549 cells. A total of 1×107 transfected A549 cells were subcutaneously injected into male BALB/c nude mice (6–7 weeks old, Beijing, People’s Republic of China). There were two mice in our each group. Tumor volumes were measured every 3 days. At 18 days after transplantation, mice were sacrificed. Tumors were harvested, weighed, photographed and frozen for RRM2, Cyclin A1, Cyclin B1, CDK2 and p-AKT expression analysis. The experimental procedures were performed following the Guidelines for Care and Use of Laboratory Animal with the approval of Ethics Committee of Luohe Central Hospital.
6
Generating Luciferase-Expressing and Osteopontin-Silenced U87-MG Cells
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U87-MG cells were first transformed using pLenti6-Luc plasmid that was generated by cloning the luciferase gene into the pLenti6/V5-D-Topo vector (K4955-10, Life Technologies). Following selection of the positive cells, the U87-MG-luc+ cells were further transformed using pLKO-shOPNi plasmid that was generated by cloning the shRNA sequence in the pLKO-puro-IPTG 3xLacO vector. The shRNA OPN vector (TRCN 0000342563) was purchased from Sigma. Lentiviral vectors used for the above transformations were generated by co-transfecting the Lenti-X 293T cells (Clontech) with pLenti6-Luc or pLKO-shOPNi. Lentiviral supernatants were collected 48h, 72h and 96h post transfection, filtrated and concentrated 100x by ultracentrifugation. The lentiviral vectors were then titrated with qPCR Lentivirus Titration Kit (LV900; Applied Biological Materials) and used to transduce U87-MG cells. Stably transduced U87-MG cells expressing luciferase were isolated and maintained in medium supplemented with 10 μg/mL of blasticidin (ant-bl-1; Invitrogen). Then, U87-MG-luc+ were transduced with shRNA-OPNi lentiviral vectors, selected and maintained in medium supplemented with 10 μg/mL of puromycin (ant-pr-1; Invitrogen) and 10 μg/mL of blasticidin.
7
Differentiation and Genetic Manipulation of 3T3-L1 Adipocytes
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The 3T3-L1 preadipocytes (fibroblasts) were cultured and differentiated into adipocytes as previously described (Zeigerer et al., 2002 (link)). The cells were originally received from G. Baldini (University of Arkansas) in 1992. These cells were last tested for mycoplasma in 2017. Experiments were performed on day 5 after differentiation. The 3T3-L1 adipocyte cell lines stably expressing shRNA sequences against RAB10 or TBC1D4 and expressing control shRNA sequences that do not target genes in the mouse genome have been described previously (Eguez et al., 2005 (link); Sano et al., 2007 (link)).
Immunoabsorption experiments were performed using 3T3-L1 cell lines stably expressing WT and mutant HA-GLUT4-GFP. To generate these cell lines, cDNA constructs encoding WT, F5Y, and F5A-HA-GLUT4-GFP (Lampson et al., 2000 (link); Blot and McGraw, 2006 (link)) were subcloned into the pLenti6/V5-D-TOPO vector (K4955-10; Life Technologies). The 293FT packaging cells were transfected with lentiviral cDNA using Lenti-X packaging system (631276; Takara). Cultured media containing lentiviral particles were harvested after 72 h and used to infect 3T3-L1 preadipocytes. HA-GLUT4-GFP-positive cells were sorted by FACS and cultured in selection medium supplemented with blasticidin (A11139-03; Invitrogen).
Immunoabsorption experiments were performed using 3T3-L1 cell lines stably expressing WT and mutant HA-GLUT4-GFP. To generate these cell lines, cDNA constructs encoding WT, F5Y, and F5A-HA-GLUT4-GFP (Lampson et al., 2000 (link); Blot and McGraw, 2006 (link)) were subcloned into the pLenti6/V5-D-TOPO vector (K4955-10; Life Technologies). The 293FT packaging cells were transfected with lentiviral cDNA using Lenti-X packaging system (631276; Takara). Cultured media containing lentiviral particles were harvested after 72 h and used to infect 3T3-L1 preadipocytes. HA-GLUT4-GFP-positive cells were sorted by FACS and cultured in selection medium supplemented with blasticidin (A11139-03; Invitrogen).
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