Construction method for model of animals with retinitis pigmentosa diseases and application

A technology for retinitis pigmentosa and degenerative diseases, applied in the field of construction of animal models of retinitis pigmentosa, can solve the problems of complex cell biochemical process, lack of animal models of retinitis pigmentosa, inconvenience of retinitis pigmentosa, etc., and achieves broad application prospects. Effect

Active Publication Date: 2018-05-15
SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

3) The pathogenic molecular mechanism is complex
The cell biochemical processes that have been found to be involved in RP pathogenic genes are very complex, such as light signal transduction, cytoskeleton, RNA splicing, ubiquitination and degradation, etc. Difficulties in therapeutic intervention
[0004] At present, there is still a lack of animal models of retinitis pigmentosa, which brings inconvenience to further in-depth and systematic research on retinitis pigmentosa.

Method used

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  • Construction method for model of animals with retinitis pigmentosa diseases and application
  • Construction method for model of animals with retinitis pigmentosa diseases and application
  • Construction method for model of animals with retinitis pigmentosa diseases and application

Examples

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Embodiment

[0049] In this example, the mouse Hkdc1 gene was knocked out by CRISPR-Cas9 technology to construct an animal model of retinitis pigmentosa disease, as follows.

[0050] 1. Design the gRNA sequence targeting knockout of the HHkdc1 gene, and obtain the F0 generation of Hkdc1 knockout mice

[0051] Design the gRNA target site sequence for the second exon region of the mouse Hkdc1 gene, as follows:

[0052] 5'-CCTGTATCACATGCGGCTCTCGG-3' (SEQ ID NO. 1).

[0053] The RNA was synthesized in vitro, microinjected into fertilized mouse eggs together with Cas9 endonuclease, and after maturation, the first founder mouse (founder) with gene mutation was obtained.

[0054] Schematic diagram of the structure of the gRNA sequence targeting the Hkdc1 gene figure 1 shown.

[0055] 2. Sequencing verification of Hkdc1 gene knockout results

[0056] Using the genomic DNA of the mouse, the fragment near the CRISPR target site was sequenced, and it was determined that the first builder mouse ha...

Embodiment 1

[0112] Using Example 1 to Construct a Hkdc1 Gene Knockout Mouse Model for Research on Retinal Degenerative Diseases

[0113] 1. Frozen sections were carried out on the retina of the knockout mouse, and analyzed using immunohistochemical staining (the immunohistochemical method is the same as in Example 1), and it was found that at the age of 9 months, the homozygous Hkdc1 gene knockout (KO) Abnormal trafficking of retinal rhodopsin (Rhodopsin) in mice: accumulation in photoreceptor cell inner nodes and cell bodies ( Figure 5 ). Rhodopsin is the main light-sensing protein, which is extremely critical to the formation of vision, and its mislocalization will lead to apoptosis of photoreceptor cells. Therefore, it can be known that Hkdc1 gene plays an important role in retinal degenerative diseases.

[0114] 2. The results of H&E staining showed the progressive apoptosis of retinal photoreceptor cells in KO mice (homozygous Hkdc1 knockout mice)

[0115] Paraffin sections and he...

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Abstract

The invention discloses a construction method for a model of animals with retinitis pigmentosa diseases and application and relates to the technical field of medical engineering. According to the construction method for the model of the animals with the retinitis pigmentosa diseases, Hkdcl gene sequences on genomes of target animals are knocked out, the model of the animals with the retinitis pigmentosa diseases is obtained, the model of the animals presents typical characteristics, such as retina function damage and gradualness apoptosis of photoreceptor cells, of retinitis pigmentosa, the model of the animals can be applied to research of the retinitis pigmentosa diseases and medicine for screening treatment of the retinitis pigmentosa diseases, and the method has wide application prospects.

Description

technical field [0001] The invention relates to the technical field of medical engineering, in particular to a construction method and application of an animal model of retinitis pigmentosa disease. Background technique [0002] Retinitis Pigmentosa (RP) is a group of highly heterogeneous degenerative retinal photoreceptor cell lesions and fatal ophthalmic diseases. The initial onset usually affects the rod cells first, resulting in decreased dark vision and impaired peripheral vision. Tubular vision develops until blindness; fundus examination reveals retinal pigmentation. The incidence of RP in my country is about 1 / 4000. Based on the huge population base in my country, there are millions of RP patients, which brings a heavy burden to families and society. Even more unfortunately, there is currently a lack of in-depth and systematic research on the etiology and pathological mechanism of the disease, and the pathogenic mechanism of quite a few RP genes is still unclear, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/85
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03C12N9/1205C12N15/85C12N2800/80C12N2810/10C12Y207/01001A01K67/0275A01K2267/0306C12N9/22C12N15/1137C12N15/907C12N2310/20C12N15/11
Inventor 朱献军杨正林张琳杨业明
Owner SICHUAN PROVINCIAL PEOPLES HOSPITAL
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