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The pgCAR sequence was inserted between the BamH I and Hind III sites of the pcDNA3.1 (+) expression vector to construct the pcDNA3.1-pgCAR expression plasmid.
PgCAR was amplified by PCR from boar liver cDNA using oligonucleotide primers designed using the sus scrofa CAR sequence information from NCBI and the cutting sites of restriction enzymes, BamH I and Hind III, were added at ends of the primers.
The pgCAR sequences were endonuclease digested by BamH I and Hind III in a double digestion reaction and visualized on 1% agarose gels and cloned into the pcDNA3.1 (+) expression vector.
Dose response of pgCAR activator phenytoin in HepG2: A dose response curve was generated to determine the proper concentration range that induces pgCAR reporter gene activation by Phenytoin using the dual-luciferase reporter assay.
Effects of the Active Ingredients of Herbal Medicines on pgCAR
The results of the pgCAR dual-luciferase assay showed at Fig.
4, we can see that pgCAR is activated by Phenytoin and the dose response experiment was conducted to determine at what concentration phenytoin could significantly activate the pgCAR receptor in HepG2 cells.
For the pgCAR dual-luciferase assay, NR1 as the CAR responsive element was cloned upstream of the firefly luciferase reporter gene to pick up the ligands which regulate pgCAR causing altered reporter gene expression.
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- PGC 42407