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Construction method and application of sdk2 gene mutant mouse model

A technology of mouse model and construction method, applied in genetic engineering, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as difficulty in obtaining ocular materials, unclear pathogenic mechanism, and restricting research development

Active Publication Date: 2022-04-19
BEIJING TONGREN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there have been no reports of congenital cataracts caused by the cell adhesion molecule SDK2 at home and abroad, the pathogenic mechanism is still unclear, and the corresponding treatment methods have not been systematically studied.
However, the ocular materials of human patients are difficult to obtain and the reproducibility is poor, which restricts the development of related research.

Method used

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  • Construction method and application of sdk2 gene mutant mouse model
  • Construction method and application of sdk2 gene mutant mouse model
  • Construction method and application of sdk2 gene mutant mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of SDK2 Gene Mutation Mouse Model

[0045] The target gene SDK2 is located on the reverse strand of chromosome 11 in the mouse genome, with a length of about 289.85kb. The Gene ID in NCBI is 237979, and a total of 6 transcripts can be formed. In this example, the transcript SDK2-001 (Transcript ID: ENSMUST00000041627) As an example, a mouse model with a mutation at the R87C site (corresponding to the R83C site of the human SDK2 gene) ("CGC" changed to "TGC") was constructed.

[0046] S1: Construction of sgRNA for gene knock-in.

[0047] S1.1: Construct sgRNA for gene knock-in in http: / / crispr.mit.edu / , and get 14 sgRNA sequences, including 7 5`Guide (SEQ ID No.1~7) and 7 3`Guide (SEQ ID No.8~14), specifically as shown in Table 1:

[0048] Table 1 Candidate sgRNA sequences

[0049]

[0050]

[0051] S1.2: Insert the above sgRNA into the Cas9 plasmid respectively to construct 14 kinds of Cas9 / sgRNA plasmids;

[0052] S1.3: Construct sequencin...

Embodiment 2

[0104] Example 2 Application of SDK2 gene mutation mouse model to study the role of cell adhesion junctions in lens development

[0105] The inventors discovered for the first time that mutations in SDK2 are associated with congenital cataracts, suggesting that cell adhesion molecules may have an important function in the development and / or maintenance of the lens. Taking the pathogenic mutation as a starting point, by comparing with the wild type, we explored the role of cell adhesion junctions in lens development and function maintenance, and elucidated the pathogenic molecular mechanism of SDK2 mutation. The specific experimental method is as follows:

[0106] (1) Observe the shape and degree of lens opacity with a slit lamp microscope, quantify the area of ​​lens opacity under a dark field microscope, and perform phenotypic evaluation;

[0107] (2) Choose 2-month-old mice as the research object, measure the weight and diameter of the removed lens, evaluate the impact of S...

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Abstract

The invention provides a method for constructing an SDK2 gene mutant mouse model. Based on the CRISPR / Cas9 system, an sgRNA capable of specifically recognizing the SDK2 gene is designed and constructed, and the sgRNA and a targeting vector constructed based on the sgRNA are injected into mouse fertilized eggs. In the middle, after embryo transfer, the F0 generation mice with SDK2 gene mutation were selected from the output mice and crossed with wild-type mice to obtain the F1 generation mouse model with SDK2 gene mutation. The mouse model constructed by this method can be stably passed down, and it is convenient to study the mechanism of action of the SDK2 gene in mouse hereditary cataract in practical applications. In the case that the research materials of human patients are not easy to obtain and are restricted by medical ethics, this application provides The mouse model will become an important tool in the research of hereditary cataract, providing a stable genetic research model in the research of pathogenic mechanism, treatment method, drug screening and cataract surgery.

Description

technical field [0001] The invention belongs to the technical field of animal model construction, in particular to a method for constructing an SDK2 gene mutation mouse model and its application. Background technique [0002] Congenital cataract is the first blinding eye disease in children. There are 200,000 children worldwide blinded by cataract, accounting for about 5-20% of children's blindness. The causes of congenital cataract are many and complex, and can be divided into three categories: genetic factors, environmental factors and unknown causes. Studies have confirmed that genetic factors are the most important pathogenic factors, accounting for about 50% of the causes. More than 40 pathogenic genes have been found to be related to autosomal dominant cataract (Cat-Map; http: / / cat-map.wustl.edu / ), including 13 crystallin genes, 7 membrane protein genes ( Gap link protein and channel protein, etc.), 6 developmental and transcription factor genes, 3 cytoskeletal protei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90A01K67/027A61D19/04A61K49/00
CPCC12N15/113C12N15/8509C12N15/907C07K14/47A01K67/0276A61D19/04A61K49/008C12N2310/20A01K2207/15A01K2217/075A01K2227/105A01K2267/03
Inventor 王开杰
Owner BEIJING TONGREN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV