Generation of pgc-1α mice with conditional knockout of GABAergic neurons

A PGC-1, gene knockout mouse technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc. Avoid the effects of decreased survival

Inactive Publication Date: 2018-06-01
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

with PGC-1α + / + and PGC-1α + / - genotype compared to PGC-1α - / - The body weight of genetic mice decreased by 10%-15% 2 months after birth (Lin JD, Wu P, Tarr PT, et al. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1α null mice. Cell, 2004. 119: 121 -135.) Decreased survival rate and weight loss will hinder the use of PGC-1α gene knockout mice to study the relationship between this gene and the mechanism of neuropsychiatric diseases

Method used

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  • Generation of pgc-1α mice with conditional knockout of GABAergic neurons
  • Generation of pgc-1α mice with conditional knockout of GABAergic neurons
  • Generation of pgc-1α mice with conditional knockout of GABAergic neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Introduction and Breeding of Conditional Gene Knockout Mice

[0042] 1.1 Experimental animals

[0043] PGC-1α flox / + Conditional knockout mouse, strain name B6.129-Ppargc1α tm2Brsp / JNju , purchased from Jackson Lab in the United States, 2 males and 2 males. Dlx5 / 6 Cre-IRES-EGFP Donated by Professor Zhao Chunjie of Southeast University, two males and two males, the mice express Cre recombinase in GABAegic interneurons; Dlx5 / 6 Cre-IRES-EGFP The transgenic mice were transformed into a 0.5kb fragment containing id6 / id5 enhancer, Beta-globin enhancer and enhanced green fluorescent fusion protein EGFP (Enhanced green fluorescent fusion protein). SPF grade C57BL / 6J mice were purchased from the Experimental Animal Center of Jiangsu University [license number: SCxk (Su) 2015-0001].

[0044] alpha flox / + Feeding of mice

[0045] PGC-1α flox / + The mice were fed in accordance with the SPF animal feeding standards. After isolation and observation, no abnormalitie...

Embodiment 2

[0046] Example 2: PGC-1α flox / + Mouse identification, population expansion and conservation

[0047] 2.1 Extraction of genomic DNA from PGC-1α conditional knockout mice

[0048] Male and female mice purchased from Jackson Lab in the United States were mated, and the offspring were numbered by cutting off the toes (cutting out the toes of the offspring 5-6 days after birth), numbering from hind limbs to forelimbs, and from right to left. Clipped toe tissue was used to extract genomic DNA. The general steps of genomic DNA extraction are as follows: (1) Put mouse toes (in the order of 1-10) into 1.5 mL EP tubes, centrifuge for 30 s to make them sink to the bottom of the tube. (2) Add 30 μL lysate (0.5% 20% Tween-20, 1M KCL 50mM, 1M MgCl 2 15 mM, 1M Tris-HCl 2.5 mM, pH8.0, add 200 μg / mL proteinase K (3 μL) before use), (3) digest at 55°C for 3-5 h, centrifuge at 12,000 rpm for 2 min, 95°C, 15 min, inactivate Proteinase K was centrifuged at 12,000 rpm for 2 minutes, and the DNA...

Embodiment 3

[0061] Example 3: Dlx5 / 6 Cre-IRES-EGFP Mouse Genotyping and Amplification

[0062] Dlx5 / 6 Cre-IRES-EGFP The mouse was transformed with a 0.5kb fragment containing the id6 / id5 enhancer, beta-globin enhancer, and enhanced green fluorescent fusion protein EGFP (Enhanced green fluorescent fusion protein). Dlx5 / 6 Cre-IRES-EGFP The offspring of mice mated with C57BL / 6J mice theoretically include Dlx5 / 6 Cre-IRES-EGFP Transgenic and wild-type mice.

[0063] For the transgenic mice expressing Cre recombinase specifically in GABAergic interneurons, due to the transfer of EGFP, 1-2 days after the birth of the offspring, the young mice were placed on a fluorescence microscope and excited by blue light. Whether the Cre recombinase is transferred into the mouse skull (the green fluorescent protein can be seen when transferred), such as Figure 4 shown, Figure 4 It can be seen in Figures A, B, and C that green fluorescence appears, indicating that these mice have been transformed ...

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Abstract

The invention relates to a GABAergic neuron conditional knockout gene PGC-1 alpha mouse model and a construction method thereof and belongs to the field of animal models and application thereof. The construction method includes the steps that a PGC-1 alpha <flox / flox> mouse is obtained in the first place; then, a PGC-1 alpha <flox / +> is mated with a C57BL / 6J, and the PGC-1 alpha <flox / +> is obtained, then the PGC-1 alpha <flox / +> and the C57BL / 6J are mated with a Dlx5 / 6<Cre-IRES-EGFP> mouse, and the full knockout D1x5 / 6< Cre-IRES-EGFP >, PGC-1 alpha <flox / flox>, semi knockout D1x5 / 6< Cre-IRES-EGFP > and a PGC-1 alpha <flox> mouse are obtained; as a result, a gene knockout mouse on GABAergic internuncial neuron conditional knockout PGC-1 alpha is prepared, and a credible animal model is provided for mechanism research on neurodevelopment and neurodegenerative diseases.

Description

technical field [0001] The invention relates to a GABAergic neuron conditional knockout gene PGC-1α mouse model and a construction method thereof, belonging to the field of animal models and applications thereof. Background technique [0002] Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), as a transcriptional coactivator, regulates mitochondrial synthesis and mitochondrial oxygen metabolism. plays a key role in gene expression. Transcriptional coactivator PGC-1α recruits other specific transcription factors, induces histone deacetylation, affects chromosome structure changes and initiates transcription (Lin J, WH., Tarr PT, Zhang CY, et al. Transcriptional co-activator PGC-1alpha drives the formation of slow-twitch muscle fibres. Nature, 2002, 418:797-801.). [0003] In peripheral tissues, PGC-1α has been defined as a metabolic regulatory target for its ability to induce mitochondrial biosynthesis and transcription of genes for antioxidant enzymes. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52
CPCA01K67/0278A01K2217/075A01K2227/105A01K2267/0318C12Q1/6888C12Q2600/16
Inventor 王佳张维宁钱进军王玉聪王春燕张颖吴丹萍方秋珏王玉王艳泓胡天媛
Owner JIANGSU UNIV
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