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Construction method and application of incoordination animal model

An animal model, ataxia technology, applied in the field of medical engineering, can solve problems such as lack of animal models

Active Publication Date: 2018-01-16
SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is a lack of related animal models that can be used for ataxia research

Method used

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  • Construction method and application of incoordination animal model
  • Construction method and application of incoordination animal model
  • Construction method and application of incoordination animal model

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Experimental program
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Effect test

Embodiment 1

[0046] In this embodiment, taking mice as the target animal, the method for constructing the animal model of ataxia provided by the present invention is described, specifically as follows.

[0047] 1. Refer to the method of the Chinese patent application No. 2017103803265, named the construction method and application of the mouse model of pancreatic beta cell conditional knockout of Tmem30a gene, to obtain the conditional knockout of Tmem30a gene (exon No. 3) homozygous small mouse;

[0048] 2. The Tmem30a gene conditional knockout homozygous mice were mated with the Pcp2-Cre gene transgenic mice to obtain the cerebellar Purkinje cell conditional knockout Tmem30a gene mice ( figure 1 A). Such as figure 1 As shown in A, Pcp2-Cre transgenic mice were mated with Tmem30a gene knockout homozygous mice, and half of the offspring carried both Pcp2-Cre and Tmem30a conditional knockout mice. The animal was mated with a Tmem30a gene knockout homozygous mouse to obtain a cerebellar P...

Embodiment 2

[0051] In order to determine the recombination efficiency of Cre-mediated loxP sites, this embodiment constructed the tdTomato reporter gene ( figure 2 ). In the absence of Cre, the STOP gene cassette with loxP recombination sites on both sides can prevent the expression of the downstream red fluorescent protein tdTomato. When Cre exists, the STOP gene box is removed in Cre-specifically expressed tissues, resulting in the expression of tdTomato, so the expression position and expression efficiency of Cre recombinase can be determined by observing the fluorescence generated by tdTomato expression. Using immunohistochemistry, it was observed that Cre was specifically expressed in cerebellar Purkinje cells in Tmem30a KO animals (e.g. image 3 shown). image 3 The middle mark refers to: GCL: Granular cell layer, granular cell layer; PCL: Purkinjecell layer, Purkinje cell layer; ML: Molecular layer, molecular layer).

[0052] Immunohistochemical method:

[0053] After the mice...

Embodiment 3

[0056] The expression of TMEM30A protein in Tmem30a KO mice was verified by western blot (WB).

[0057] Western blot method:

[0058] ①Separate retinal tissues from wild-type and mutant mice, place them in 1.5ml centrifuge tubes, and add 200μl protein lysate RIPA;

[0059] ② After ultrasonically disrupting the retinal tissue, lyse it on ice for 20 minutes;

[0060] ③ After centrifugation at 16,000 g for 10 min at 4°C, transfer the supernatant to another clean centrifuge tube, add 50 μl protein sample solution, mix well and heat at 95°C for 5 min;

[0061] ④ After the samples are cooled, take 20 μl respectively and conduct polyacrylamide gel electrophoresis (SDS-PAGE) at 160V to separate proteins;

[0062] ⑤ After the SDS-PAGE is finished, cut the nitrocellulose membrane of appropriate size according to the needs, spread the filter paper, glue, nitrocellulose membrane and filter paper in order, and drive away the air bubbles, put the transfer membrane tank in the ice water ba...

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Abstract

The invention discloses a construction method and application of an incoordination animal model and relates to the technical field of medical engineering. The construction method of the incoordinationanimal model, disclosed by the invention, comprises the step of knocking out a target sequence on a genome in a cerebellar purkinje cell of a target animal, wherein the target sequence is a Tmem30a gene. According to the method disclosed by the invention, a Tmem30a gene sequence on the genome in the cerebellar purkinje cell of the target animal is specifically knocked out for the first time and acerebellum incoordination animal model can be constructed; the animal model has typical cerebellum incoordination characteristics: unstable treads, cerebellar atrophy, progressive apoptosis of the cerebellar purkinje cell and the like. The model can be used for researching cerebellum incoordination and provides a foundation for finding disease-causing gene of the cerebellum incoordination and exploring pathogenesis.

Description

technical field [0001] The invention relates to the technical field of medical engineering, in particular to a construction method and application of an ataxia animal model. Background technique [0002] Ataxia is a pathological state in which patients cannot maintain fine gait and complete precise movements in a certain form. Any lesion involving the afferent or efferent pathway of the cerebellum may lead to ataxia. Most of them are caused by genetic factors, so they are collectively referred to as hereditary ataxia (HA). HA is a group of genetic degenerative diseases characterized by chronic progressive cerebellar ataxia; the genetic background passed down from generation to generation, the manifestation of ataxia, and the pathological changes dominated by cerebellar damage are the three major characteristics. The classification of hereditary ataxia is still unclear, and there are more than 60 kinds reported so far, but there is no unified and recognized classification me...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85A01K67/027
CPCA01K67/027C12N15/85C12N15/90
Inventor 朱献军鲁芳杨业明张琳李姝锦杨牧张姗姗
Owner SICHUAN PROVINCIAL PEOPLES HOSPITAL
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