The 3’ untranslated region (UTR) sequence of SP1 or PARP containing the predicted mo-miR-7a binding sites was amplified from MCM genome and sub cloned into the multiple cloning sites in the downstream of the luciferase gene in the GV126 vector (Promega Corporation, Durham, NC, USA), resulting in the wild-type luciferase reporter construct, GV126-SP1-3’UTR-WT or GV126-PARP-3’UTR-WT . The mutant types, GV126-SP1-3’UTR-MU and GV126-PARP-3’UTR-MU , with the corresponding mutant seed sequence, were synthesized by site-directed mutagenesis [28 ]. These GV126 constructs expressing firefly luciferase and pRL-cmv vectors expressing Renilla luciferase (Genechem) were co-transfected into cells using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega Corporation), with Renilla luciferase activity as an internal control.
Gv126 vector
Manufactured by Promega
Sourced in United States
The GV126 vector is a plasmid designed for gene expression in mammalian cells. It features a cytomegalovirus (CMV) promoter for high-level transcription and a neomycin resistance gene for selection of stably transfected cells.
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Lab products found in correlation
2 protocols using gv126 vector
1
Luciferase Assay for miRNA Binding
2
Validating miR-133a Interaction with DAPK2 3'UTR
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To predict targets with significant change, three algorithms (TargetScan, PicTar and microRNA.org) were used for rno-miR-133a. The 3ʹUTR of Death Associated Protein Kinase 2 (DAPK2) was predicted to interact with rno-miR-133a and therefore this region was amplified by real-time (RT)-PCR and cloned downstream of luciferase gene in GV126 vector (Promega, Madison, WI, USA). Site-directed mutagenesis of rno-miR-133a target-site was carried out to abolish miR-133a binding in 3ʹ UTR of DAPK2 and pRL-TK (Promega) was used as internal control for dual luciferase assays. HEK293 cells were transfected with miR-133a mimics, along with mutant DAPK2 reporter vector (containing mutant DAPK2 3ʹ UTR), the negative control (NC) and pRL-TK control vector, as needed within the experimental setup. Luciferase activities were assayed with Dual-Luciferase Reporter Assay kit (Promega). All procedures were performed on the basis of the manufacturer's instructions.
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