For application of c-Met by polymerase chain reaction (PCR), a pair of primers were designed and synthesized as follows: Forward primer: 5’-GAGGATCCCCGGGTACCGGTCGCCACCATGAAGGCTCCCACCGCGCTGGCACCTGG-3’; Reverse primer: 5’-TCCTTGTAGTCCATACCTGTGTTCGCTTCGCCGTCAATGTTGTCTTG-3’. The resulting c-Met cDNA was inserted into the lentiviral vector GV358 (sequence elements: Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin; Shanghai Genechem Company, China) to create the lentiviral vector, GV358-c-Met. We cotransfected the recombinant and two lentiviral helper plasmids (Helper1.0 and Helper2.0; Shanghai Genechem Company, China) into 293 T cells to generate the target lentivirus with an infectious viral titer of 2 × 108 TU/mL, which was measured using a fluorescence assay method. In parallel, the control lentivirus was produced by cotransfecting the lenti-GFP empty vector GV358 with GFP but without c-Met, and two lentiviral helper plasmids (Helper1.0 and Helper2.0) into 293 T cells, and an infectious viral titer of 1 × 108 TU/mL was obtained.
Gv358
Manufactured by Genechem
Sourced in China
The GV358 is a versatile laboratory equipment used for various scientific applications. It is designed to perform precise and consistent measurements. The core function of the GV358 is to provide accurate and reliable data.
Automatically generated - may contain errors
Lab products found in correlation
Gv248, by Genechem (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Phelper 1, by Genechem (2 mentions)
Ubi mcs 3flag sv40 egfp ires puromycin, by Genechem (1 mentions)
Helper 1, by Genechem (1 mentions)
Helper 2, by Genechem (1 mentions)
Recombinant lentiviral vector, by Genechem (1 mentions)
Recombinant lentivirus, by Genechem (1 mentions)
Hu6 mcs ubiquitin egfp ires puromycin, by Genechem (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mgc 803, by Genechem (1 mentions)
Hgc 27, by Genechem (1 mentions)
Sgc 7901, by Shanghai Cell Bank (1 mentions)
Ges 1, by Shanghai Cell Bank (1 mentions)
Mkn 45, by Shanghai Cell Bank (1 mentions)
Bgc 823, by Shanghai Cell Bank (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Phelper 2.0 vector, by Genechem (1 mentions)
Polybrene, by Merck Group (1 mentions)
26 protocols using gv358
1
Lentiviral Vector Construction for c-Met Expression
2
Overexpression of PPARγ and ERRα in EC cells
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Full-length cDNA plasmids for PPARγ (PPARG, NM_015869) and ERRα (ESRRA, NM_004451) were purchased from Genechem (Shanghai, People's Republic of China) and cloned into a lentivirus-based vector carrying the green fluorescent protein (GFP) gene (GV358, Genechem, Shanghai, China) with the following component sequence: Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin. EC cell lines transfected with the lentiviral vector were used as negative control (NC) groups, and cell lines without lentivirus treatment were used as blank controls. The lentiviral vector constructs carrying PPARγ, ERRα and NC were used to infect EC cells at a multiplicity of infection (MOI) of 100. After 72 h of infection, GFP expression was detected to calculate the infection efficiency. Cells were harvested at an infection rate >90%.
3
CYP2E1 Overexpression in MGC-803 Cells
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The CYP2E1 construct was synthesized and integrated into a recombinant lentiviral vector GV358 (Shanghai GeneChemCo., Ltd.). CYP2E1 overexpression was accomplished with the recombinant lentiviral vector (Shanghai GeneChem Co., Ltd.), and the empty vector was used as a control. MGC-803 cells were seeded into 96-well plates (5×103 cells/well in 100 µl complete medium), and three infection gradients (MOI=10, MOI=20, MOI=30) were used to infect the cells using FuGENE® 6 transfection reagent (Nanjing KeyGen Biotech Co., Ltd). The MGC-803 cells were incubated for 24 and 48 h after transfection for subsequent experiments.
4
Lentiviral Manipulation of Nrf2 in SKPs
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Recombinant lentiviruses used for overexpressing and silencing nrf2 were purchased from GeneChem (Shanghai, China). LentiORF lentiviral particles (GV358, Ubi-MCS-3FLAG-SV40-ERFP-IRES-puromycin, LVKL25280-2) and lenti-shRNA vectors (GV248, hU6-MCS-ubiquitin-EGFP-IRES-puromycin, LVpFU-GW-007) were constructed, packed, and purified by GeneChem (Shanghai, China). SKPs were transfected with lentivirus-containing lenti-RFP control (lenti-C or lenti-C-SKPs), lenti-overexpressed nrf2 (lenti-nrf2 or nrf2-over-SKPs), lenti-silenced nrf2 (lenti-shRNA-nrf2, lenti-shnrf2, or si-nrf2-SKPs), and lenti-shRNA-GFP-negative control (lenti-shNC or lenti-shNC-SKPs). Next, SKPs were incubated at 37°C/5% CO2 and selected using puromycin. Lastly, puromycin-resistant cells were allowed to grow in the SKP medium for further experiments.
5
Lentiviral Overexpression and Knockdown
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The lentivirus-based vectors for ISL2 overexpression, U2AF2 overexpression, cARF1 overexpression, RNAi-mediated knockdown of ISL2, U2AF2 and cARF1, and their negative controls were all constructed by Gene-Chem (GV358, Shanghai, China). The detailed sequence of the lentivirus-based vectors can be obtained on the GeneChem website (http://www.genechem.com.cn/index/supports/zaiti_info.html?id=50 ). The miR-342–3p mimic, inhibitor, and their negative controls were obtained from Thermo Fisher Scientific (Assay ID: MH12328 and MC12328; Thermo Fisher Scientific, Waltham, MA, USA). The sequences of all siRNAs are listed in Table S3 . The lentivirus transfection and efficacy measurements were performed as previously described [23 (link)].
6
Establishing Stable CTCF-Expressing Cell Lines
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MCF-7, MDA-MB-231, and 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO). SKBR3 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (GIBCO). All media were supplemented with 10% fetal bovine serum (FBS, HyClone). The human epithelial breast cell line, MCF-10A, was maintained in DMEM containing 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 20 ng/mL human epidermal growth factor (EGF), and 5% heat-inactivated horse serum. All cell lines were obtained from the Cell Resource Center, Peking Union Medical College. Cell lines were free of mycoplasma contamination, according to PCR analysis. The species of origin was confirmed using PCR, and the identity of the cell lines was authenticated using short tandem repeat (STR) profiling (FBI, CODIS). The full-length CTCF coding sequence was synthesized chemically and subcloned into lentivirus vector GV358 (GeneChem, Shanghai, China). MDA-MB-231 cells stably expressing CTCF and control cells were established by lentivirus-mediated gene transfer.
7
Gastric Cancer Cell Line Manipulation
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Human gastric cancer cell lines AGS, NCL-N87, MGC803 and HGC-27 were purchased from GeneChem (Shanghai, China). The human gastric cancer cell lines (BGC823, SGC7901 and MKN45) and the normal gastric cell line GES-1 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The siRNAs listed in Table S1 were designed and synthesized by Genepharma (Shanghai, China). Briefly, GC cell lines were seeded into 6-well plates and grown overnight. The next day, when the cell plating density reached 20%-30%, GC cells were transfected with siRNAs (final concentration, 50 nM) by Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. The lentiviruses for knockdown of ELF3-AS1 (C06003) were purchased from Genepharma. The lentiviruses for overexpression of SNAI1 and SNAI2 (GV358, Ubiquitin promotor) were purchased from GeneChem. Transfection was performed according to the manufacturer’s instructions. At the indicated time points, the cells were harvested for mRNA and protein analysis as well as for other assays.
8
Lentiviral Transduction of miR-874-3p and HDAC1
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miR-874-3p mimic non-targeting negative control (NC; 5'-ACUACUGAGUGACAGUAGA-3') or miR-874-3p mimic (5'-CUGCCCUGGCCCGAGGGACCGA-3') were inserted into the GV309 vector (Shanghai GeneChem Co., Ltd.); miR-874-3p inhibitor non-targeting NC (5'-CAGUACUUUUGUGUAGUACAA-3') or miR-874-3p inhibitor (5'-UCGGUCCCUCGGGCCAGGGCAG-3') were inserted into the GV280 vector (Shanghai GeneChem Co., Ltd.). For HDAC1 overexpression, the coding sequence of HDAC1 was inserted into the GV358 vector (Shanghai GeneChem Co., Ltd.). The empty vector was used as the NC for HDAC1 overexpression. 293T cells (American Type Culture Collection) were transfected with 20 µg the GV vector (GV309, GV280 or GV358) together with 15 µg pHelper 1.0 and 10 µg pHelper 2.0 vectors (both from Shanghai GeneChem Co., Ltd.) and then cultured at 37˚C in a humidified incubator in an atmosphere of 5% CO2 for 48 h to produce lentiviral particles. Transduction was performed when cells were growing exponentially and were 70-80% confluent. Polybrene (8 µg/ml, Sigma-Aldrich; Merck KGaA) and appropriate lentiviral particles (108 TU/ml, 5 µl) were added to cells for 16 h at 37˚C in a humidified incubator in an atmosphere of 5% CO2. The medium containing lentiviral particles were removed from wells and fresh medium were added. Subsequent experiments were performed 24 h later.
9
Overexpression of STAG3 in BEL-7404 and Huh-7 Cells
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For construction of the overexpression plasmids of STAG3, GV358 (GeneChem, Shanghai, China) was selected as the vector, and cDNA for STAG3 was obtained from human BEL-7404 and Huh-7 cells and then subjected to PCR amplification with the following specific primers: 5′- GAG GAT CCC CGG GTA CCG GTC GCC ACC ATG TCT TCC CCG TTG CAA AGA GCT GTG G-3′ (forward) and 5′- TCC TTG TAG TCC ATA CCG GTG AAA TCC TCA ATA TCC AGC TCT GTA GAA TC-3′ (reverse).
BEL-7404 and Huh-7 cells were prepared as cell suspensions in serum-free DMEM at a density of 1 × 105 cells/ml. These cells were infected with the STAG3 overexpression lentivirus and its negative control (NC), and the resulting infected cells served as the STAG3-overexpression (OE) and NC groups, respectively. After 6 h of incubation, the residual serum-free DMEM in each well was replaced by DMEM containing 10% FBS. The cells from each group were incubated for 72 h, and the efficiency of infection was examined by western blot analysis.
BEL-7404 and Huh-7 cells were prepared as cell suspensions in serum-free DMEM at a density of 1 × 105 cells/ml. These cells were infected with the STAG3 overexpression lentivirus and its negative control (NC), and the resulting infected cells served as the STAG3-overexpression (OE) and NC groups, respectively. After 6 h of incubation, the residual serum-free DMEM in each well was replaced by DMEM containing 10% FBS. The cells from each group were incubated for 72 h, and the efficiency of infection was examined by western blot analysis.
10
Construction of IRF7 Expression Lentiviral Vector
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To construct the IRF7 expression vector, the fragments encompassing the IRF7 sequence were synthetized chemically, and then cloned into the BamHI and AgeI sites in lentiviral vector (GV358) (Genechem, Shanghai). Plasmid DNA was transformed in MAX Efficiency® DH5a™ Competent Cells (Invitrogen, Mount Waverley, Victoria, Australia) as described by the manufacturer. Positive colonies were further cultured for plasmid amplification and purification using the Wizard® Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer's instructions. Purified plasmid DNA was verified for the presence of IRF7 insert by restriction digest with BamHI/AgeI. All constructs were further confirmed by DNA sequencing analysis.
293T cells were cultured at 10cm culture dishes for 24h, before the cells were co-transfected with GV358-IRF7 DNA (20μg), pHelper 1.0 (15μg), and pHelper 2.0 (15μg) using Lipofectamine 2000 in 1ml volume for 6hr (Invitrogen). After co-transfection for 6hr, the transfected supernatants were removed, and the dished were washed once by using 10ml 1×PBS, and added new cell medium. The culture supernatants were collected after the 293T cells cultured for 48hr and used as virus stock after concentration. Viral titer was determined by HIV-1 P24 Antigen ELISA (ZeptoMetrix Corporation) after infection.
293T cells were cultured at 10cm culture dishes for 24h
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