Recombinant plasmid containing OATP1B1 promoter and reporter gene and method for screening OATP1B1 inducer
A reporter gene and recombinant plasmid technology, applied in the field of plasmids containing fluorescent reporter genes, can solve the problems of rarely used, human hepatocyte model ethics, and tissue source culture conditions can not achieve high throughput, etc., to achieve easy materials, The effect of fast detection speed and high sensitivity
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[0029] Example 1
[0030] Construct the OATP1B1 promoter fluorescent reporter gene plasmid and transiently transfect it into HepG2 cells to construct a dual fluorescent reporter gene technology platform.
[0031] 1. Material
[0032] 1. Plasmids, cell lines
[0033] pGL-4.17 fluorescent reporter gene vector, pPEM-T vector, pcDNA3.1-myc / hisB(-) eukaryotic expression vector: American promega company.
[0034] HepG2 cell line: Tumor Cell Bank of Chinese Academy of Medical Sciences.
[0035] 2. Main reagents
[0036] Fetal bovine serum (Hangzhou Sijiqing); MEM medium (Gibco, USA); 50ml cell culture flask, 24-well cell culture plate (Corning, USA); RevertAid TM First Strand cDNA reverse transcription kit (Fermentas); Lipofectamine TM 2000 liposomes (Invitrogen, USA); DNA extraction kit (promega); Trizol (Invitrogen, USA).
[0037] 3. Main instruments
[0038] PCR machine (Thermo, USA); electrophoresis equipment (Beijing Liuyi); CO 2 Cell incubator (U.S. ThermoForm, SeriesII); ultra-clean wor...
Example Embodiment
[0157] Example 2
[0158] The experimental method established above was used to study whether baicalin, a monomer compound of traditional Chinese medicine, can induce the expression of OATP1B1 by activating PXR.
[0159] Steps:
[0160] 1. Culture HepG2 cells;
[0161] When HepG2 cells grow to log phase, digest to 2×10 per well 5 The density of each cell is seeded on a 24-well plate, and the cells are grown to 80%.
[0162] 2. Co-transfection
[0163] 2.1 Each well needs to be transferred into 600ng PXR plasmid, 300ng OATP1B1 promoter fluorescent reporter gene plasmid and 50ng sea cucumber luciferase. After diluting DNA and Lipofectamine 2000 liposomes with an appropriate amount of culture medium without antibiotics and fetal bovine serum, they were mixed and placed at room temperature for 20 minutes.
[0164] 2.2 Wash the cells three times with PBS, and then add 1 ml of MEM medium without fetal bovine serum to each well.
[0165] 2.3 Add 200ul of the above mixture (DNA / liposome) per well...
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