Construction method and application of SDK2 gene mutation mice mice

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: 2020-10-16
BEIJING TONGREN HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 m...

Method used

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  • Construction method and application of SDK2 gene mutation mice mice
  • Construction method and application of SDK2 gene mutation mice mice
  • Construction method and application of SDK2 gene mutation mice mice

Examples

Experimental program
Comparison scheme
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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 of the mouse genome, with a length of about 289.85kb. The Gene ID in NCBI is 237979, and 6 kinds of transcripts can be formed. The transcript SDK2-001 (Transcript ID: ENSMUST00000041627) As an example, construct a mouse model of mutation ("CGC" becomes "TGC") at the R87C site (corresponding to the R83C site of the human SDK2 gene).

[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 a total of 14 sgRNA sequences, including 7 5`Guide (SEQ ID No.1~7) and 7 3 `Guide (SEQ ID No. 8-14), as shown in Table 1:

[0048] Table 1 Candidate sgRNA sequence

[0049]

[0050]

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

[0052] S1.3: Construction of sequencing primers for target fragments, as shown in Table 2:

[0053]...

Embodiment 2

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

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

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

[0107] (2) Two-month-old mice were selected as the research object, and the weight and diameter of the removed lens were measured to evaluate ...

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Abstract

The invention provides a construction method of an SDK2 gene mutation mice model. The mice model comprises the following steps of designing and constructing sgRNA capable of specifically identifying an SDK2 gene on the basis of a CRISPR/Cas9 system, injecting the sgRNA and a targeting vector constructed on the basis of the sgRNA into a mice fertilized egg; and after embryo transplantation, screening out F0-generation mice with SDK2 gene mutation from the output mice, and hybridizing the F0-generation mice with wild type mice to obtain an F1-generation mice model with SDK2 gene mutation. The mice model constructed by the method can be stably passaged, the action mechanism of the SDK2 gene in mice hereditary cataract can be conveniently researched in practical application, and under the condition that human patient research materials are not easy to obtain and are restricted by medical ethics, the SDK2 gene can be stably cloned. The mice model provided by the invention can become an important tool in the research of hereditary cataract, and a research model capable of realizing stable heredity is provided in the research of pathogenesis, treatment methods, drug screening, cataract surgery and the like.

Description

Technical field [0001] The invention belongs to the technical field of animal model construction, and in particular relates 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. Globally, 200,000 children are blinded by cataract, which accounts for about 5-20% of children’s blindness. Congenital cataracts have many and complex causes, which can be divided into three categories: genetic factors, environmental factors and unknown causes. Studies have confirmed that genetic factors are the most important pathogenic factor, accounting for about 50% of the disease. More than 40 pathogenic genes have been found to be related to autosomal dominant cataract (Cat-Map; http: / / cat-map.wustl.edu / ), including 13 lens protein genes and 7 membrane protein genes ( Gap link protein and channel protein, etc.), 6 developmental and transcription factor genes, 3 cytoskeletal...

Claims

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Application Information

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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
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