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Construction and application of GIPR reporter gene stably transfected cell strain

A reporter gene and cell line technology, applied in the fields of application, animal cells, genetic engineering, etc., can solve the problems of insufficient sensitivity, high time cost, and incomplete characterization of GIPR activation/antagonism mechanism

Pending Publication Date: 2022-03-25
宁波熙宁检测技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a GIPR reporter gene stably transfected cell line construction and application, to solve the biological activity measurement method, biological analysis quantitative method and neutralizing antibody detection method in the GIPR agonist / antagonist drug development process, the detection steps are cumbersome and time-consuming High cost, high cost of reagent consumables, complex interpretation of experimental data, insufficient sensitivity of the method, poor anti-interference ability, inability to fully characterize the mechanism and effect of GIPR activation / antagonism, lack of versatility, etc., the inventor of the present application constructed the GIPR report Gene stably transfected cell lines, and use the cell lines to develop biological activity assay methods not limited to GIPR agonist drugs, bioanalytical quantitative methods and neutralizing antibody assay methods

Method used

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  • Construction and application of GIPR reporter gene stably transfected cell strain
  • Construction and application of GIPR reporter gene stably transfected cell strain
  • Construction and application of GIPR reporter gene stably transfected cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] CRE reporter gene stably transfected cell line construction process

[0029] 4μg CRE reporter gene plasmid (customized by Sangon, containing CRE binding region, minipromoter SEQ ID NO.: 2 and luciferase gene) and 4ul X-tremeGENE HP DNA transfection reagent (Roche-06366244001) using opti-MEM medium (Thermo 31985 -070) 100ul mixed evenly, after standing at room temperature for half an hour, add 1ml DMEM60% confluent 293T cells into a single well of a 6-well plate, change the cell medium on the second day, and transfer to a T25 cell culture flask on the third day Subculture and add 0.5μg / ml puromycin DMEM medium for pressurized culture. Most of the cells died, and after maintaining 0.5 μg / ml puromycin DMEM for three passages, the cells were monoclonal plated in 96-well plates at 1 cell / well, 200ul / well. After 14 days of monoclonal plating in a 96-well plate, observe the cell condition in each well under a microscope, and select the monoclonal into a 24-well plate. After 1...

Embodiment 2

[0033] Evaluation of CRE reporter gene transfection efficiency

[0034] Take the 293T cells transfected with the reporter gene expression plasmid, pressurize them with 1 μg / ml puromycin DMEM for two generations, and take 80uL of the digested cell suspension (10000cells / well) into each well of a white opaque 96-well plate , add 20 ul of Forskolin with a concentration of 150 uM or 20 ul of medium, mix evenly, put into 37oC 5% CO2 incubator and incubate for 5 hours. Add 100ul Bright-Glo TM Luciferase Assay reagent (Promega E2620), mixed at room temperature at 1000 rpm for 5 minutes, put into a chemiluminescence detector for detection.

[0035] See the test results figure 1 , After the signal value of Forskolin treatment was compared with the medium-treated background group, the transfected cells had a strong signal response, and the signal-to-noise ratio was greater than 50 times. The experimental results showed that the CRE reporter gene had been inserted into 293T cells.

Embodiment 3

[0037] Functional evaluation of CRE reporter gene monoclonal cell line in 24-well plate

[0038] Select a single clone from the 96-well plate plated by the monoclonal plate and transfer it to a 24-well plate. After the clones in the 24-well plate are basically full, digest the cells with 100ul trypsin for 2-3 minutes, and then add 400ul medium to resuspend the cells. Take 320uL of digested cell suspension (10000cells / well) into 3 wells of a white opaque 96-well plate, add 20ul of Forskolin with a concentration of 150uM or 20ul of medium respectively, mix well, and place in 37oC 5% CO2 Incubate for 5 hours in the incubator. Add 100ulBright-Glo TM The Luciferase Assay reagent was mixed at room temperature at 1000rpm for 5 minutes, and put into a chemiluminescence detector for detection.

[0039] The detection results of the monoclonal are shown in the table below. The experimental results show that different monoclonals are treated with culture medium or Forskolin, resulting ...

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Abstract

The invention discloses construction of a GIPR reporter gene stably transfected cell strain, which comprises the following steps: transfecting 293T cells by using CRE reporter gene plasmids, adding eukaryotic antibiotics for screening and monoclonal selection, and performing functional evaluation and selection to obtain an appropriate CRE reporter gene stably transfected cell strain monoclonal; gIPR lentivirus is used for infecting CRE reporter gene stably transfected cell strain monoclonal cells, eukaryotic antibiotics are added for screening and monoclonal selection, and the appropriate GIPR reporter gene stably transfected cell strain is obtained through functional evaluation. A biological activity determination method, a biological analysis quantitative method and a neutralizing antibody determination method which are not limited to GIPR agonist drugs are developed by utilizing the cell strain. The problems that the detection steps are tedious, the time cost is high, the reagent consumable cost is high, experimental data interpretation is complex, the method is not sensitive enough, the anti-interference capacity is poor, the GIPR activation / antagonism mechanism and effect cannot be completely represented, and universality is not achieved are solved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a cell line stably transfected with a GIPR reporter gene; and a method for measuring GIPR receptor agonist activity using the cell line, and a quantitative method for GIPR receptor agonist using the cell line , the detection method of the neutralizing antibody of GIPR receptor agonist / antagonist, the present invention further relates to the method that utilizes this cell line to the activity assay of the antagonist of GIPR receptor, utilizes this cell line to the antagonism of GIPR receptor Dosage Quantification Method. Background technique [0002] GIPR (UniProt ID: P48546) is a receptor for gastric inhibitory polypeptide (GIP), a member of the G protein-coupled receptor family. Gastric inhibitory polypeptide and glucagon (GLP-1) are both intestinal hormones, which are secreted by endocrine L cells and K cells respectively, and have the function of promoting digestion. GIP...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867C12Q1/66G01N33/74G01N33/68C12Q1/02
CPCC07K14/723C12N15/86C12N5/0686C12Q1/66G01N33/5044G01N33/74G01N33/6854C12N2510/00C12N2740/15043G01N2333/726G01N2500/10C12N2503/02
Inventor 黄启宽朱国振余跃云杨雨生
Owner 宁波熙宁检测技术有限公司
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