GCGR reporter gene stably transfected cell strain as well as construction method and application thereof

A reporter gene and construction method technology, applied in applications, animal cells, genetic engineering, etc., can solve the problems of poor anti-interference ability, incomplete characterization, and high cost of reagents and consumables

Pending Publication Date: 2021-09-03
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

[0008] The object of the present invention is to provide a GCGR reporter gene stably transfected cell line and its construction method and application, which can solve one or more of the above-mentioned problems in the prior art, such as cumbersome detection steps, high cost of time, high cost of reagent consumables, and experimental data Interpretation is complicated, the method is not sensitive enough, the anti-interference ability is poor, the mechanism and effect of GCGR activation / antagonism cannot be fully characterized, and it is not universal, etc.

Method used

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  • GCGR reporter gene stably transfected cell strain as well as construction method and application thereof
  • GCGR reporter gene stably transfected cell strain as well as construction method and application thereof
  • GCGR reporter gene stably transfected cell strain as well as construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] CRE reporter gene stably transfected cell line construction process

[0035] 4μg CRE reporter gene plasmid (customized by Sangon, containing CRE binding region, minipromoter SEQ ID NO.: 1 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.

[0036] 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...

Embodiment 2

[0040] Evaluation of CRE reporter gene transfection efficiency

[0041] 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 20ul of Forskolin with a concentration of 150uM or 20ul of medium, mix well, put in 37°C 5% CO 2 Incubate for 5 hours in the incubator. 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.

[0042] 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

[0044] Functional evaluation of CRE reporter gene monoclonal cell line in 24-well plate

[0045] 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 culture medium, mix well, and place in 37°C 5% CO 2 Incubate for 5 hours in the incubator. Add 100ul Bright-Glo TM The Luciferase Assay reagent was mixed at room temperature at 1000rpm for 5 minutes, and put into a chemiluminescence detector for detection.

[0046] The detection results of the monoclonal are shown in Table 1 below. The experimental results show that different monoclonals are treated with culture medium or Forskolin, resulting in ve...

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Abstract

The invention discloses a GCGR reporter gene stably transfected cell strain and a construction method and application thereof, a cell membrane of the cell strain expresses a GCGR receptor, the cell strain comprises polynucleotide, the polynucleotide has a CRE-bound binding sequence and is connected with a downstream promoter, the promoter is connected with an open reading frame, and the reading frame expresses a reporter gene protein. The inventor constructs a GCGR reporter gene stably transfected cell strain, and develops a biological activity determination method, a biological analysis quantitative method and a neutralizing antibody determination method which are not limited to GCGR agonist and antagonist drugs by using 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 GCGR activation/antagonism mechanism and effect cannot be completely represented, and is lack of universality.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a construction method and application of a GCGR reporter gene stably transfected cell line. Background technique [0002] GCGR (UniProt ID: P47871) is a receptor for glucagon, a member of the G protein-coupled receptor family, which plays a key role in regulating blood sugar levels and maintaining blood sugar homeostasis effect. GCGR exerts physiological functions after being activated by glucagon. Glucagon has a strong effect of promoting glycogenolysis and gluconeogenesis, which can significantly increase blood sugar. 1mol / L hormone can make 3×106mol / L glucose Rapidly decomposed from glycogen. Glucagon activates the phosphorylase of liver cells through the cAMP-PK system and accelerates the decomposition of glycogen. In the GCGR signaling pathway, glucagon activates intracellular signals by binding to GCGR on the cell membrane surface, leading to the activation of intrac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/85C12N15/65C12N15/12C12N5/10C12Q1/02G01N33/68
CPCC12N15/867C12N15/85C12N15/65C07K14/723C12N5/0686G01N33/5008G01N33/6854C12N2740/15043C12N2800/107C12N2510/00C12N2503/02G01N2500/10G01N2333/726
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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