Knock-in non-human mammal model with uncoupling protein 1-luciferase gene and its construction method and application

A non-human mammal and uncoupling protein technology, applied in the field of non-human mammal model and its construction, can solve the problems of time-consuming, labor-intensive, labor-intensive and expensive, and achieve simple results

Active Publication Date: 2015-09-30
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, no matter whether real-time quantitative RT-PCR or western blot is used for drug screening, it is time-consuming, labor-intensive and expensive.

Method used

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  • Knock-in non-human mammal model with uncoupling protein 1-luciferase gene and its construction method and application
  • Knock-in non-human mammal model with uncoupling protein 1-luciferase gene and its construction method and application
  • Knock-in non-human mammal model with uncoupling protein 1-luciferase gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Knock-in method for constructing a mouse model with uncoupling protein 1-luciferase

[0029] Such as figure 1 As shown, it is a construction strategy map of the mouse model knocked in with UCP1-luciferase in this embodiment, including the following steps:

[0030] 1. Remove the stop codon of UCP1

[0031] UCP1 has 6 exons, and the last exon of UCP1, that is, the stop codon after the 6th exon, is removed;

[0032] 2. Link luciferase gene

[0033] The tandem peptide 2A (SEQ ID NO: 1, whose purpose is to allow the luciferase gene to be translated into a protein together with UCP1, but not fused with the UCP1 protein, so it will not affect the activity of the UCP1 protein) is removed in step 1 The back of the last exon of UCP1 of the stop codon is connected into the luciferase gene; the sequence after the connection of the luciferase gene is shown in SEQ ID NO:2;

[0034] 3. Obtain the mouse model

[0035] A mouse model with UCP1-luciferase knock-in was const...

Embodiment 2

[0036] Example 2 Detection of luciferase signals in various tissues of the mouse model knocked in with uncoupling protein 1-luciferase in Example 1

[0037] Include the following steps:

[0038] 1) Take a knock-in uncoupling protein 1-luciferase mouse obtained in Example 1, kill it by neck dislocation, and separate each tissue;

[0039] 2) Aliquot 20mg of each tissue into a 1.5mL centrifuge tube, add 200uL luciferase signal detection buffer (150mM KCl, 20mM HEPES, 5mM Mgcl 2 , 1 mM EGTA. NaOH to adjust the pH to 7.0), and grind the tissue into a homogenate;

[0040] 3) Centrifuge at a low temperature of 4°C and 13kpm for 20 minutes;

[0041] 4) Take 15uL of supernatant, 15uL and 30μL of high-sugar DMEM medium Reagent ( Luciferase Assay System, Promega) mixed with CulturPlate TM -96 in the wells of a white solid bottom 96-well plate (PerkinElmer);

[0042] 5) Shake for 10 minutes;

[0043] 6) Measure on a fluorescent light meter (the luciferase reacts with the substra...

Embodiment 3

[0045] Example 3 The mouse model knocked in with uncoupling protein 1-luciferase in Example 1 is detected by luciferase signal of adipocytes differentiated from adipose stem cells of inguinal white adipose tissue under the stimulation of drugs FGF21 and AM580

[0046] Include the following steps:

[0047] 1. Isolation of primary adipose mesenchymal cells from inguinal white fat

[0048] 1) Take a knock-in uncoupling protein 1-luciferase mouse obtained in Example 1, kill it by neck dislocation, soak it in 75% alcohol for 5 minutes, then transfer it to the operating table, and take out the white groin Fat;

[0049] 2) Wash 3 times with PBS, cut up the adipose tissue with scissors, add 10ml collagenase I solution (prepared with D-Hanks solution, add 0.1g collagenase I to 100ml) for 1g adipose tissue, and place it at 37°C for 40 minutes;

[0050] 3) Filter with a 250 μm filter membrane, add cell culture medium, then transfer to a centrifuge tube, and centrifuge at 1000 rpm for 3...

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Abstract

The invention discloses a non-human mammal model with an input UCP1-1cuiferase (uncoupling protein 1-1cuiferase) gene as well as a construction method and the application of the non-human mammal model. The constructed non-human mammal model with the input UCP1-1cuiferase gene can be taken as a screening platform of white fat cell brownness drugs, and the simple, rapid and large-scale screening on drugs for regulating and controlling the expression of the UCP1 can be realized; on the premise that non-human mammal is not hurt, the expression and distribution of the UCP1 can be directly subjected to bio-assay by utilizing a fluorescent imager.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a non-human mammal model knocked in with an uncoupling protein 1-luciferase gene, its construction method and application. Background technique [0002] It has long been thought that the main function of fat cells is to store energy. However, with the in-depth study of fat cells, other functions of fat cells have gradually emerged. Studies have shown that adipocytes play an important role in metabolic diseases such as immune response, hypertension, obesity, and insulin resistance. [0003] Initially, fat cells were divided into two types: white fat cells and brown fat cells. However, with the discovery of beige (brown-in-white, brite) cells in the inguinal fat of rodents, brown adipocytes can be subdivided into two types: classic brown adipocytes and brite / beige adipocytes [0004] White adipocytes contain a large, round lipid droplet with a flattene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01K67/027C12N15/85
CPCA01K67/027C12N15/85
Inventor 吴东海聂涛毛刘峰李快唐小凤惠晓艳刘彭涛徐爱民
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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