pGL3-Basic Vector

Reporter gene assay luciferase - vascular smooth muscle cells (VSMC)

Experiment
Reporter gene assay luciferase - vascular smooth muscle cells (VSMC)
Product
pGL3-Basic Vector from Promega
Manufacturer
Promega

Protocol tips

Protocol tips
Transfect for 48h and harvest cells.

Publication protocol

VSMCs and 293T cells were seeded on 6-well plates at a density of 6×105 cells/cm2 and cultured overnight in a humidified atmosphere with 5% CO2 at 37°C. Cells were then transfected with 20 nM miR-17 mimic or 20 nM inhibitor or 20 nM control miRNA (Guangzhou RiboBio Co., Ltd., Guangzhou, China) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 24 h, cells were collected for western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses.

To construct the pGL3-RB-3′ untranslated region (3′UTR), the full length 3′UTR of the human RB mRNA was cloned into the pGL3-control vector (Promega Corp., Madison, WI, USA). To construct the miR-17 promoter reporter genes, the DNA fragment upstream of the transcription start site of the miR-17 gene was inserted into the pGL3-basic vector (Promega Corp.). For luciferase reporter assays, 293T cells were transiently transfected with miR-17 mimics and Renilla-luciferase reporter plasmids (Promega Corp.) containing the pGL3-RB-3′UTR (with the binding region: AGGCGCUU) or pGL3-RB-3′UTR MUT (with the mutant binding region: UUCGUGAA) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, cells were collected and reporter gene activity was determined using the dual-luciferase assay system (Invitrogen; Thermo Fisher Scientific, Inc.). Renilla luciferase activity was used for normalization of transfection efficiency. Reporter luciferase activity assay was also used to assess the function of NF-κB p65 regulating miR-17 promoter activity. There were three NF-κB p65 binding sites in the regulatory sequence of the miR-17 gene.

Full paper   Login or join for free to view the full paper.

Reviews

pGL3-Basic Vector from Promega has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Discussion

2 years ago

Author: J.Han China

Which transfection reagent would work better on stem cells?

I am planning on running reporter assays on stem cells and I would like to know which transfection reagent would work better?

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Reporter gene assay luciferase - vascular smooth muscle cells (VSMC) using pGL3-Basic Vector from Promega.

View full paper   Login or join for free to view the full paper.

Manufacturer protocol

Download the product protocol from Promega for pGL3-Basic Vector below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Reporter gene assay luciferase - vascular smooth muscle cells (VSMC) using pGL3-Basic Vector from Promega. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Become shareholder Discussions About us Contact Privacy Terms