A full-length human VNN1 cDNA was cloned into a lentiviral vector (Shanghai GeneChem Co., Ltd.) for constitutive gene expression. The lentiviral vector was co-transfected with packaging vectors (Shanghai GeneChem Co., Ltd.) into 293T cells (cat. no. GNHu17; from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences). The PANC-1 and CFPAC-1 cells were infected with empty or VNN1-expressing lentiviruses. The PANC-1 and CFPAC-1 cells with a stable overexpression of VNN1 were termed as PV and CV, and the PANC-1 and CFPAC-1 cells transfected with the empty vector were referred to as PE and CE.
Lentiviral vector
Manufactured by Genechem
Sourced in China
Lentiviral vectors are a type of viral vector used for the delivery of genetic material into target cells. They are derived from the human immunodeficiency virus (HIV) and are designed to efficiently transduce both dividing and non-dividing cells. Lentiviral vectors can be used to introduce genes, knockdown gene expression, or modify the genome of target cells.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions)
4 1bb cd3ζ expression cassette, by Genechem (2 mentions)
Scrambled shrna, by Genechem (2 mentions)
Mimic nc, by RiboBio (2 mentions)
Puromycin, by Beyotime (2 mentions)
Plvx puro, by Takara Bio (2 mentions)
Polybrene, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Hep3b, by American Type Culture Collection (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Smmc 7721, by American Type Culture Collection (1 mentions)
Sk hep1, by American Type Culture Collection (1 mentions)
Gv248 lentiviral vector, by Genechem (1 mentions)
Gv492, by Genechem (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
82 protocols using lentiviral vector
1
Lentiviral-mediated VNN1 overexpression
2
Lentiviral Vector Construction for ZFAS1 Study
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The lentiviral vector containing ZFAS1 DNA sequence (ZFAS1‐wt), the lentiviral vector containing mutated (predicted miR‐296‐5p binding sites) ZFAS1 DNA sequence (ZFAS1‐mut), the lentiviral vector containing ZFAS1 shRNA (shZFAS1) and the negative control were purchased from Shanghai Genechem company. To generate pmirGLO‐ZFAS1‐wt and pmirGLO‐USF1‐wt, ZFAS1 cDNA and USF1 3′UTR were amplified by PCR and then subcloned into pmirGLO plasmid (Generay Biotechnology). The pmirGLO‐ZFAS1‐mut and pmirGLO‐USF1‐mut were generated by the site‐directed mutagenesis kit (Stratagene). The constructs were validated by DNA sequencing.
3
Lentiviral Manipulation of H19 Expression
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A lentiviral vector with H19 knockdown and overexpression and a lentiviral vector alone, which was used as a negative control, were constructed by Genechem (Shanghai, China). Cells with H19 knockdown were defined as shH19 group and the corresponding control group was called shCon. The nucleotide sequences of the three pairs of double-stranded shRNA fragments were based on the sequences of the H19 gene to construct an interfere vector and included the following: shH19-1: 5′-CCGGCAGCCTTCAAGCATTCCATTACTCGAGTTTTTG-3′; shH19-2: 5′-CCGGCAGGAGAGTTAGCAAAGGTGACTCGAGTTTTTG-3′; and shH19-3: 5′-CCGGAACCCACAACATGAAAGAAATCTCGAGTTTTTG-3′, the scramble sequence of shCon was 5′-CCGGTTCTCCGAACGTGTCACGTTTTTTG-3′. Cells that overexpressed H19 were defined as the PH19 group and the corresponding control group was called Pvector. Cells of the HTR-8 were seeded in six-well plates at 40% confluence on the day before transfection. HTR-8 cells were transfected at the proper multiplicity of infection. Green fluorescent protein expression was used to assess the infection efficiency after 96 h of infection. RT-qPCR was performed to evaluate the efficiency of H19 expression.
4
Lentiviral-mediated Uba2 Modulation
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For Uba2 knock-down, a lentiviral vector encoding a short interfering (si)RNA targeted against Uba2, and a negative control lentiviral vector (Control; NC) were constructed (Genechem, Shanghai, China). For over-expression studies, the Uba2 gene was synthesized according to the human UBA2 mRNA sequence and inserted into a lentiviral vector (Genechem). Transfection was carried out as previously described[15 (link)]. BGC-823 cells were infected with siUba2 or siNC lentivirus at a multiplicity of infection of 100. SGC-7901 cells were infected with lentivirus-Uba2 (Uba2) or lentiviral empty vector (EV) at a multiplicity of infection of 10. The infected cells were harvested and used for experimentation after 72 h.
5
Overexpression and Silencing of circRNA_0008532 and MTGR1
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Human circRNA_0008532 and MTGR1 cDNA was amplified by PCR and cloned into a lentiviral vector (GeneChem, Shanghai, China). Oligos of circ_0008532 and MTGR1 shRNAs were synthesized and inserted into a lentiviral vector (GeneChem, Shanghai, China). Stable cell lines were selected for 10 days with 0.5 mg/ml puromycin. The miR-155-5p\miR-330-5p mimics, negative control, and anti-miR-155-5p\anti-miR-330-5p inhibitor were purchased from RiboBio (Guangzhou, China). MiRNA or miRNA inhibitor was transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.
6
Knockdown of MAZ in Liver Cancer Cells
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SK-Hep-1, HepG2, Hep3B, SMMC-7721 and Huh-7 cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS). Bel-7402, Li-7 and L02 cells were cultured in RPMI-1640 medium with 10% FBS. All the cell lines were grown at 37°C in a 5% CO2/95% air atmosphere.
5 shRNAs that targeted MAZ and a shRNA control were constructed in a lentiviral vector and purchased from Genechem (Shanghai, China). The shRNAs were transferred to SMMC-7721 cells and we chose the 3# and 4# of shMAZ (target sequence: GCCCTTCAAATGTGAGAAA and GGCCATGTTCCCGGTGTTT) with best knock-down effect for the follow-up analyses.
5 shRNAs that targeted MAZ and a shRNA control were constructed in a lentiviral vector and purchased from Genechem (Shanghai, China). The shRNAs were transferred to SMMC-7721 cells and we chose the 3# and 4# of shMAZ (target sequence: GCCCTTCAAATGTGAGAAA and GGCCATGTTCCCGGTGTTT) with best knock-down effect for the follow-up analyses.
7
Engineered GD2/CAR Lentiviral Vector
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The chimeric GD2/CAR is composed of GD2 scFv and a 4-1BB-CD3ζ expression cassette that was designed and synthesized by the GeneChem Biotechnology Company (Shanghai, China), as shown in Fig. 3 a. The GD2 scFv was derived from a high-affinity 14.G2a monoclonal antibody. The 4-1BB-CD3ζ expression cassette contains the hinge and transmembrane (TM) region of CD8α. GD2 scFv and 4-1BB-CD3ζ were connected in-frame by overlap PCR. The generated GD2/CAR was verified by DNA sequencing and cloned into the BamHI sites of a lentiviral vector (Genechem Biotechnology, China); the resultant product was named GD2.BBζ CAR. The specific structure of the viral vector is shown in Additional file 1 : Figure S1. The intracellular domain of the CARs has the self-cleaving 2A peptide connected to an EGFP green fluorescent label. The sequences of all PCR primers are available upon request.
8
Lentiviral POSTN Gene Transfer Protocol
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The lentiviral vector carrying the POSTN gene and the negative control lentiviral vector (GV248), as well as that with POSTN-shRNA and its corresponding negative control (GV492), were obtained from Genechem Co., Ltd. (Shanghai, China). The infection was conducted according to the manufacturer's instructions. The efficiency of lentiviral infection was determined by real-time PCR and western blotting, and stably transfected clones were obtained through puromycin screening.
9
Modulating miR-216a and DNMT1 in PMVECs
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The PMVECs were seeded into 6-well plates until they reached 70–80% confluence. According to the manufacturer’s instructions, we used lipofectamine 3000 (Invitrogen, CA, USA) transfected miR-216a mimic, NC mimic (RiboBio Corporation, Guangzhou, China) into PMVECs for 24 h and then the PMVECs were treated with 2.5% CSE for 24 h. The PMVECs were seeded into a six-well plate and when the density reached 30–40%, an MOI value of 100 was used, virus infection enhancement solution, overexpression of DNA methyltransferase 1 (DNMT1) (lentivirus-DNMT1) and lentiviral vector (Genechem Corporation, Shanghai, China) were added; the solution was changed after 16 h of infection and the viral infection was observed under a fluorescence microscope after 72 h. Solutions were used for subsequent experiments.
10
Knockdown of CBX3 Gene in U373 Cells
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The lentiviral vector containing CBX3 siRNA was synthesized by Genechem (Shanghai, China). siRNA target sequences (shCBX3-1, 5′-ACGTGTAGTGAATGGGAAA-3′ and shCBX3-2, 5′-TGAAGAATTTGTCGTGGAA-3′) for the CBX3 gene (NM_016587) were designed, and a nonsilencing siRNA sequence (5′-TTCTCCGAACGTGTCACGT-3′) was adopted as a negative control (NC, shCtrl).
U373 cells were seeded in six-well culture plates and transfected with lentivirus according to the manufacturer’s instructions (MOI = 5). The culture medium was replaced after 10 h, and mCherry expression was observed under a fluorescence microscope (Olympus) 3 days after infection.
U373 cells were seeded in six-well culture plates and transfected with lentivirus according to the manufacturer’s instructions (MOI = 5). The culture medium was replaced after 10 h, and mCherry expression was observed under a fluorescence microscope (Olympus) 3 days after infection.
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