Preparation method of SD rat T cell-deficient genetic model

A genetic model and cell technology, applied in the field of genetic engineering and genetic modification, can solve problems such as frameshift mutation, double-strand DNA break, gene knockout, etc., and achieve high originality, high cutting efficiency, and convenient follow-up detection.

Inactive Publication Date: 2017-12-08
XINXIANG MEDICAL UNIV
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Problems solved by technology

The CRISPR-Cas9 system is mainly composed of three parts, namely Cas9 protein, precursor CRISPR RNA (pre-crRNA) and trans-activating crRNA (tracrRNA). CRISPR-Cas9 recognizes a specific DNA sequence and cuts at a specific site to form a double Strand DNA breaks, and in the absence of a template, non-homologous recombination occurs, resulting in frameshift mutations and gene knockouts

Method used

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  • Preparation method of SD rat T cell-deficient genetic model
  • Preparation method of SD rat T cell-deficient genetic model
  • Preparation method of SD rat T cell-deficient genetic model

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Embodiment 1

[0028] 1. According to the principle of CRISPR / Cas9 system, using CRISPOR online design software (http: / / crispor.tefor.net / crispor.cgi), in the target sites of rat Lck gene exon3 (exon ID: ENSRNOE00000091030) and exon4 (exon ID: ENSRNOE00000091075) selects one specific site as the target sequence of sgRNA, the two target sequences are: sgRNA1 (SEQ ID NO.1): sgRNA1: 5'-GCCCAAGTCTCCATCATGGGAGG-3'; sgRNA2 (SEQ ID NO.1) NO.2): 5-GACCCACTGGTCACCTATGAGGG-3.

[0029] 2. Order 3 primers from Shanghai Bailiger Biotechnology Co., Ltd.: Rat-LCK-IVT-1 (SEQ ID NO.3): 5'-TTAATACGAC TCACTATAGG GGCCCAAGTC TCCATCATGG GGTTTTAGAG CTAGAAATAGCAAG-3', Rat-LCK-IVT-2 (SEQ ID NO. 4): 5'-TTAATACGAC TCACTATAGG GGACCCACTGGTCACCTATG AGTTTTAGAG CTAGAAATAG CAAG-3', Rat-LCK-IVT-3 (SEQ ID NO. 5): 5'-AAAAAAGCAC CGACTCGGTG CC-3'.

[0030]3. Obtain double-stranded DNA with T7 promoter by PCR amplification (add T7 promoter sequence to the 5' end of the synthesized primer sequence, and then obtain double-stranded...

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Abstract

The invention relates to a preparation method of an SD rat T cell-deficient genetic model, and belongs to the technical fields of genetic engineering and genetic modification. Specific knockout on a key gene Lck, which is in charge of controlling development of rat T cells, is achieved in a rat by virtue of a CRISPR-Cas9 system, so that the SD rat T cell-deficient genetic model is obtained. According to the preparation method provided by the invention, two specific targeting sites, which are targeted to the rat Lck gene, are determined for the first time, and experiments prove that the specific targeting sites are quite high in shear efficiency; two sgRNAs are simultaneously adopted to conduct targeting on the rat Lck gene, and the large-fragment deficient gene knockout rat is obtained, so that in one aspect, the targeted gent completely loses functions, and in the other aspect, subsequent detection is also facilitated. The constructed T cell-deficient SD rat animal model is great significance to immunity and disease researches.

Description

technical field [0001] The invention relates to a preparation method of a genetic model of SD rat T cell deletion, belonging to the technical field of genetic engineering and genetic modification. Background technique [0002] The activation of Lck (Lymphocyte-specific protein tyrosine kinase) is a key factor in initiating the TCR signaling pathway, which plays a key role in the development of the thymus and the differentiation and development of T cells, so the Lck gene was knocked out in rats. , that is, a rat animal model of T cell deletion can be obtained. Currently, there are three commonly used gene knockout methods: 1. Zinc finger nucleases (ZFNs); 2. Transcription activator-like effector nucleases (TALENs); 3. CRISPR-Cas9. The possible prospects of zinc finger nuclease ZFNs in the site-specific transformation of plant genomes, but due to the difficult synthesis and assembly technology of ZFNs, it is difficult to implement in general laboratories, and ZFNs are prone ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22A01K67/027
Inventor 张黎琛卢燎勋梁银明黄蓉晁天柱郑前前罗静谷妍蓉袁鹏
Owner XINXIANG MEDICAL UNIV
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