Method for constructing mouse model with autistic spectrum disorder

A mouse model, autism technology, applied to other methods of inserting foreign genetic material, using microinjection method, and cells modified by introducing foreign genetic material, etc., can solve the problem of unproven and established direct causality. , to achieve high accuracy and low off-target effect

Active Publication Date: 2018-12-07
SHANGHAI TECH UNIV
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Problems solved by technology

However, a direct causal relationship between methylation of the M

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  • Method for constructing mouse model with autistic spectrum disorder
  • Method for constructing mouse model with autistic spectrum disorder
  • Method for constructing mouse model with autistic spectrum disorder

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Embodiment 1

[0088] 1. Construction of site-directed methylation system plasmid

[0089] 设计正向引物带有NheI酶切位点及15bp骨架载体同源臂GGGAGACCCAAGCTGGCTAGCACCATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGCGGCAGCATGTTCGAAACCGTGCCTGTG(SEQ ID NO:1),反向引物带有15bp同源臂CCTCTTCTCAGCTGGGTGGCTGCCGCGGGGCACTAGTCCGCTGCTGAAGCTGCGCCCGCTGCTTGAAAAATACTTGAAATATTCT(SEQ ID NO:2),加水溶解至10μM。 The Novizan high-fidelity enzyme kit (Vazyme, p501-d2) was used to amplify human DNMT3L cDNA (source obtained by reverse transcription with a reverse transcription kit (Takara, DRR036A), template concentration: 1 ng / μl). The forward primer CCAGCTGAGAAGAGGAAGCCC (SEQ ID NO: 3) was designed, the reaction primer had a 15bp homology arm TAGAGTATTTCTTGTCGCTCTCGGGGGTGGCGCTCTCGCTGGTACCGGGGGTCTCGCTGCCGCT (SEQ ID NO: 4), and dissolved in water to 10 μM. Human DNMT3A cDNA was amplified using Novizan High Fidelity Enzyme Kit (Vazyme, p501-d2) (the source was obtained by reverse transcription with a reverse transcription kit (Takara, DRR036A), template concentrati...

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Abstract

The invention provides a method for constructing a mouse model with the autistic spectrum disorder. The method is characterized by comprising the step of injecting a methylation carrier and a gRNA carrier targeting an MeCP2 gene TSS region into cytoplasm by virtue of an embryo microinjection technique in the oosperm period of a mouse, so as to obtain the mouse model with the autistic spectrum disorder, wherein the methylation carrier contains a dCas9 fragment, a human source DNMT3L and a DNMT3A catalytic function domain. By virtue of the fixed point methylation carrier, the specific methylation of loci in cells can be effectively realized.

Description

technical field [0001] The invention relates to a site-specific methylation technology in mice, which belongs to the field of epigenetic editing, and more specifically relates to the fusion of CRISPR system and methyltransferase, combined with embryonic microinjection technology for in vivo MeCP2 Gene-specific methylation to create a mouse model of autism spectrum disorder. This technology can also be used to simulate and construct mouse models of physiological state changes caused by abnormal methylation in the development of other genes, and to interfere with diseases caused by abnormal epigenetic modifications in vivo. Background technique [0002] Autism spectrum disorders (ASD) are a complex group of neurological disorders that affect 1.5% of newborn children. People with ASD have symptoms such as social impairment, anxiety, and repetitive stereotyped behaviors. ASD has a strong genetic component and clinical heterogeneity. More than 400 genes have been shown to be a...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/85C12N5/10A01K67/027
CPCA01K67/0275C12N15/85C12N15/89A01K2267/0318A01K2227/105A01K2217/07A01K2207/15
Inventor 陆宗阳刘真黄行许
Owner SHANGHAI TECH UNIV
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