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Construction and application of Pparg gene site-directed mutagenesis mouse model

A gene site-directed mutation, mouse model technology, applied in the field of genetic engineering

Pending Publication Date: 2022-06-07
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there are many gaps in the development of Pparg gene site-directed mutation mice

Method used

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  • Construction and application of Pparg gene site-directed mutagenesis mouse model
  • Construction and application of Pparg gene site-directed mutagenesis mouse model
  • Construction and application of Pparg gene site-directed mutagenesis mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction method of mouse model of Pparg gene site-directed mutation, the principle is as follows figure 1 As shown, it specifically includes the following steps:

[0044] 1. Design of CRISPR / Cas9 gRNA for Pparg gene:

[0045] According to the Pparg gene sequence and the mutations T166A (ACC→GCT) and T166D (ACC→GAT) to be introduced, two gRNA sequences were designed. For the specific sequences, see SEQ ID NO.1 (T166A mutation) and NO.2 (T166D mutation) in the sequence table. )

[0046] 2. Construction of Donor vector:

[0047] The Donor vector construction method is to first artificially synthesize target sequence oligonucleotide primers with different restriction enzyme cleavage site recognition sequences at the 5′ end, and directly anneal the two pairs of primers by PCR to synthesize target sequence DNA carrying different sticky ends. A short fragment was inserted into the vector to construct a Donor vector (CRISPR / Cas9 targeting vector) targeting the...

Embodiment 2

[0061] Example 2: 8-week-old TA and TD mice were subjected to adipose tissue sectioning and H&E staining. By analyzing the morphological characteristics of adipose tissue, it was found that adipocytes in the adipose tissue of TA mice were smaller and more numerous, and single adipocytes accumulated lipids; conversely, adipocytes in TD mice were larger and less numerous, and individual adipocytes accumulated more lipids ( image 3 .A); Simultaneously, using fluorescence quantitative PCR to analyze the changes of metabolism-related genes in adipose tissue, it can be found that the adipose tissue of TA mice is characterized by high fatty acid metabolism, while the fatty acid catabolism of TD mice is blocked ( image 3 .B). Subsequently, the two point mutant mice were fed high-fat for 3 months, and the liver and muscle tissues were isolated to analyze their pathological status. steatosis ( Figure 4 .A-B). Simultaneous analysis of insulin sensitivity, substance and energy metab...

Embodiment 3

[0062] Example 3: point mutant mice and wild-type mice were fed with high-fat (60% fat content) for 3 months. Subsequently, the visceral fat of the mice was isolated and paraffin sectioned, and the tissue sections were analyzed by H&E pathological staining; the results showed that a large number of inflammatory immune cells were infiltrated in the visceral adipose tissue of wild-type mice, and the adipose tissue presented a state of chronic inflammation , while the visceral adipose tissue of TA mice contained less immune cell infiltration ( Figure 5 .A). At the same time, sorting mouse primary macrophages and performing quantitative PCR analysis showed that TA mutations reduced the classical activation of macrophages (M1 polarization) and enhanced the alternative activation phenotype of macrophages (M2 polarization). ). Conversely, TD mutations reduce macrophage alternative activation phenotype (M2 polarization) and instead enhance classical activation (M1 polarization) ( ...

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Abstract

The invention belongs to the technical field of gene engineering, and relates to construction and application of two Pparg gene site-directed mutagenesis mouse models. According to the invention, a CRISPR / Cas9 gene editing technology is used to carry out artificial mutation on the position of the Pparg gene for coding threonine at the 166th site, and thus two site-directed mutagenesis mouse strains are successfully manufactured. On the basis of the obtained Pparg gene site-directed mutagenesis mouse strains, various physiological and pathological states of the two strains are analyzed through various cell, biochemical and molecular biological research means, and the application value of the Pparg gene site-directed mutagenesis mouse strains in related research is determined. Meanwhile, the Pparg gene site-directed mutagenesis mouse can be applied to screening of related drugs, drug design, pharmacology / toxicology, pharmacodynamics, pharmacokinetics and exploration of therapeutic targets.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the construction and application of two mouse models of Pparg gene site-directed mutation. Background technique [0002] PPARγ is a nuclear receptor transcription factor that plays an important role in many biological processes such as cell proliferation, differentiation, metabolism, and functional phenotype maintenance. PPARγ exists in a variety of cell types, such as adipocytes, liver cells, muscle cells and other metabolic organ-derived cells; macrophages, T / B cells, dendritic cells and other immune cells; neurons and other nervous system-derived cells . As a classic clinical drug target, PPARγ can significantly enhance insulin sensitivity and reduce blood sugar in patients with type II diabetes when agonized by full agonists thiazolidinediones (TZDs). At the same time, this agonistic effect can reduce the infiltration of inflammatory cells in metabolically disorde...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02C12N15/113C12N15/85C12N15/10C12N15/12
CPCA01K67/02C12N15/1136C12N15/8509C12N15/102C07K14/475A61K49/0008C12N2310/20C12N2800/107A01K2227/105A01K2267/03A01K2217/05
Inventor 沈萍萍杨南飞
Owner NANJING UNIV
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