For Gli1 overexpression, the full-length Gli1 sequence was synthesized by cloning the Gli1 gene into the GV146 vector (Shanghai GeneChem Co., Ltd.) via XhoI and EcoRI sites, and was referred to as pGli1-IRES-EGFP. Empty GV146 vector acted as a negative control (NC) of pGli1-IRES-EGFP and was referred to as pIRES-EGFP. For knockdown of Gli1, short hairpin RNA (shRNA) directed against Gli1 was ligated into the GV102 vector (Shanghai GeneChem Co., Ltd.) and was referred to as sh-Gli1; a non-targeting sequence was ligated into the GV102 vector as the NC of sh-Gli1, and was referred to as sh-NC. The target sequence was 5′-CCTCTGTCTACTCACCACA-3′ and the NC sequence was 5′-TTCTCCGAACGTGTCACGT-3′. Eca109 and Eca109R cells were seeded in 6-well plates at a density of 6×105 cells/well one day before transfection. Briefly, 5 µl Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and 2.5 µg plasmid were diluted into 125 µl Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and mixed for 5 min at room temperature. Cells were then incubated with this solution, and complete medium was added to the final volume of 2 ml. Cell transfection was performed for 24 h following the manufacturer's protocols prior to further experiments.
Gv146 vector
Manufactured by Genechem
The GV146 vector is a plasmid-based expression system designed for recombinant protein production. It contains a strong promoter and multiple cloning sites to facilitate the insertion of target genes. The vector is commonly used in various molecular biology applications, including gene expression studies and protein engineering.
Automatically generated - may contain errors
2 protocols using gv146 vector
1
Overexpression and Knockdown of Gli1 in Esophageal Cancer
2
PRDM16 Overexpression and Knockdown in Thyroid Cancer Cells
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The PRDM16 sequence was synthesized and subcloned into a GV146 vector (GENECHEM Co., Ltd., Shanghai, China). PC overexpression plasmid was bought from GENECHEM Co., Ltd. (Shanghai, China), and the GV492 vector was used as a negative control. PC-siRNAs were designed and synthesized by Biotend Co., Ltd. (Shanghai, China). The sequences of PC-siRNAs are listed in Supplementary Table 2 . The cells were incubated for 48 h before use in assays. Human pGL3-PC-promoter and four truncated segments of pGL3-PC-promoters for the luciferase assay were constructed by GENEWIZ Co., Ltd. (Suzhou, China). Plasmids were transfected into cells using Hieff Trans Liposomal Transfection Reagent (Yeasen, Shanghai, China) according to the manufacturer’s protocol. We adopted the qRT-PCR assay and Western blotting to evaluate overexpression of PRDM16. PC overexpression and knockdown efficiency were tested using qRT-PCR. BCPAPPRDM16–OE and BCPAPPRDM16–NC cell lines were filtrated from BCPAP cells transfected with PRDM16 overexpression plasmids or the negative control vector using 200 μg/ml neomycin and cultured with RPMI-1640 with 100 μg/ml neomycin. The stable cell lines were identified using fluorescence microscope for green fluorescence to ensure the transfection efficiency was more than 90%.
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