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GABAergic neuron conditional knockout gene PGC-1 alpha mouse model and construction method thereof

A PGC-1 and mouse model technology, applied in biochemical equipment and methods, microbe measurement/inspection, animal husbandry, etc., can solve the problems of decreased survival rate and obstruction, and achieve the effect of avoiding the decline of survival rate

Inactive Publication Date: 2016-06-22
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 - / - Two months after birth, the body weight of genetic mice decreased by 10%-15% (LinJD, WuP, TarrPT, et al. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1αnullmice. Cell, 2004.119: 121-135.) The survival rate decreased, and these unfavorable factors such as weight loss would hinder the utilization of PGC-1α Gene Knockout Mice to Study the Relationship between the Gene and the Mechanism of Neuropsychiatric Diseases

Method used

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  • GABAergic neuron conditional knockout gene PGC-1 alpha mouse model and construction method thereof
  • GABAergic neuron conditional knockout gene PGC-1 alpha mouse model and construction method thereof
  • GABAergic neuron conditional knockout gene PGC-1 alpha mouse model and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Introduction and Breeding of Conditional Gene Knockout Mice

[0041] 1.1 Experimental animals

[0042] PGC-1α flox / + Conditional knockout mouse, strain name B6.129-Ppargc1α tm2Brsp / JNju , purchased from JacksonLab, USA, 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 [permit number: SCxk (Su) 2015-0001].

[0043] alpha flox / + Feeding of mice

[0044] PGC-1α flox / + The mice were fed in accordance with the SPF animal feeding standards. After isolation and observation, no abnormalities were found, they ...

Embodiment 2

[0045] Example 2: PGC-1α flox / + Mouse identification, population expansion and conservation

[0046] 2.1 Extraction of genomic DNA from PGC-1α conditional knockout mice

[0047] Male and female mice purchased from JacksonLab in the United States were mated, and the offspring were numbered by cutting off the toes (cutting out the toes of 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) Take mouse toes (according to the order of 1-10) and put them into 1.5mLEP tubes, and centrifuge them for 30s to make them sink to the bottom of the tubes. (2) Add 30μL lysate (0.5%20%Tween-20, 1MKCL50mM, 1MMgCl 2 15mM, 1MTris-HCl2.5mM, pH8.0, add 200μg / mL proteinase K before use (3μL), (3) digest at 55°C for 3-5h, centrifuge at 12,000rpm for 2min, 95°C, 15min, inactivate proteinase K, centrifuge at 12,000rpm After 2 minutes, the D...

Embodiment 3

[0060] Example 3: Dlx5 / 6 Cre-IRES-EGFP Mouse genotyping and amplification

[0061] Dlx5 / 6 Cre-IRES-EGFP The 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). Dlx5 / 6 Cre-IRES-EGFP The offspring obtained after mating mice with C57BL / 6J mice theoretically include Dlx5 / 6 Cre-IRES-EGFP Transgenic mice and wild-type mice.

[0062] For transgenic mice specifically expressing Cre recombinase in GABAergic interneurons, because EGFP was transferred, the pups were placed on a fluorescent microscope 1-2 days after birth, and excited by blue light, small cells could be seen through the skull. Whether the Cre recombinase is transferred into the mouse brain (the green fluorescent protein can be seen after the transfer), such as Figure 4 as shown, Figure 4 It can be seen in Figures A, B, and C that green fluorescence appears, indicating that these...

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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 (peroxisome proliferator-activated receptor-γcoactivator-1α, PGC-1α) as a transcriptional coactivator regulates the synthesis of mitochondria and the expression of genes related to oxygen metabolism in mitochondria plays a key role. The transcriptional co-activator PGC-1α recruits other specific transcription factors, induces histone deacetylation, affects chromosome structure changes and initiates transcription (LinJ, WH., TarrPT, ZhangCY, 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 is defined as a metabolic regulatory target for its ability to induce mit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12Q1/68
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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