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A construction method and application of an ataxia animal model

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

Active Publication Date: 2018-09-21
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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  • A construction method and application of an ataxia animal model
  • A construction method and application of an ataxia animal model
  • A construction method and application of an ataxia animal model

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Experimental program
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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. According to the method of the Chinese patent application No. 2017103803265, named as the construction method and application of the mouse model of islet beta cell conditional knockout of Tmem30a gene, 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). like 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. This animal was mated with a Tmem30a gene knockout homozygous mouse to obtain a cerebellar Purkinje ce...

Embodiment 2

[0051] In order to determine the recombination efficiency of the Cre-mediated loxP site, this example 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 blotting 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 wate...

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Abstract

Provided is a method for constructing an ataxia animal model, which comprises knocking out a target sequence on a genome of the cerebellar Purkinje cells of a target animal; the target sequence is the Tmem30a gene. Also provided are an animal model of the cerebellar ataxia constructed by the described method and a method for screening a medicament used for preventing or treating an ataxia disease using the animal model.

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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Patent Type & Authority Patents(China)
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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