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Construction method for taurine transporter gene knockout rat model based on CRISPR/Cas9 technology

A gene knockout, rat model technology, applied in the field of bioengineering, can solve the problem of no literature report on pathophysiological research

Active Publication Date: 2019-09-10
天津市环湖医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only about TauT gene knockout (TauT - / - ) mouse model, there is no literature report on the rat model knocking out the gene and its related pathophysiological research

Method used

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  • Construction method for taurine transporter gene knockout rat model based on CRISPR/Cas9 technology
  • Construction method for taurine transporter gene knockout rat model based on CRISPR/Cas9 technology
  • Construction method for taurine transporter gene knockout rat model based on CRISPR/Cas9 technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Materials and methods

[0033] 1.1 Experimental animals

[0034] The rats required for the experiment were SPF grade SD rats, weighing 240-260 g, purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0035] 1.2 Method

[0036] 1.2.1 Knockout of taurine transporter gene (TauT - / - ) Construction and breeding of SD rats

[0037] Taut - / - The SD rat model uses CRISPR / Cas9 gene editing technology to select two different sgRNA targets for the fifth exon of the TauT gene (Slc6a6), and synthesizes two pairs of oligonucleotide chains.

[0038] Among them, the two pairs of oligonucleotide chains are:

[0039] Rat-Slc6a6-gRNA1-up:TAGGCCCCTTTGTCCCACAGAC, its nucleotide sequence is shown in SEQ ID NO:1

[0040] and Rat-Slc6a6-gRNA1-down: AAACGTCTGTGGGACAAAGGGG, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0041] Rat-Slc6a6-gRNA2-up:TAGGGACAGACCCTGTCTCTGG, its nucleotide sequence is shown in SEQ ID NO:3

[0042] and Rat-Slc6a6-gRNA2-down:AAACCCAGAGACAG...

Embodiment 2

[0083] 2. Results

[0084] 2.1 Construction and reproduction of rats

[0085] Using CRISPR / Cas9 system to knock out the target gene, the Slc6a6 gene undergoes a frameshift mutation, and a stop codon is generated in advance, so that the translation is terminated early, resulting in protein dysfunction, such as figure 1 shown.

[0086] After about 15 months, 23 litters were bred from the F1 and F2 generations, and the total number of F1-F3 generations was 186. Homozygosity appeared in the F3 generation, and the homozygosity rate of this generation was about 20.59%.

[0087] F3 Generation TauT + / + : TauT + / - : TauT - / - The ratio is about 1:2.57:1.28, approximately satisfying the Mendelian law of inheritance. During the breeding process, 14 adult mice died, the mortality rate was about 7.53%.

[0088] See Table 2 for the reproduction status of the F1-F3 generation rats, and the F2 generation TauT in multiple groups + / - See Table 3 for the statistics of rat self-breeding.

...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to a construction method for a taurine transporter gene knockout rat model based on a CRISPR / Cas9 technology. For a 5th exonof a TauT gene, two sgRNA effect targets are selected, two pairs of oligonucleotide chains are synthesized, and are connected to a pUC57-sgRNA expression vector recovered by BsaI digestion, an sgRNAexpression vector is obtained by construction, Cas9mRNA and sgRNA are obtained by in vitro transcription, are injected into a male nucleus and cytoplasm of SD rat fertilized eggs, and are implanted into a pseudopregnant rat body, and the TauT gene knockout rat model is obtained by breeding. The construction method provides a reliable and stable genetic engineering model for further studying the effect of taurine on a rat neurological disease model, provides a stable animal model for studying the effect of the taurine on nervous system diseases, and lays a foundation for clarifying work such asa therapeutic effect of the taurine on related diseases.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for constructing a taurine transporter gene knockout rat model based on CRISPR / Cas9 technology. Background technique [0002] Taurine (Tau) is a sulfur-containing amino acid that can regulate cell osmotic pressure, prevent intracellular calcium overload, reduce free radical production, improve cell membrane permeability, and prevent cell swelling and deformation. An important nutrient factor during the period, it can enhance synaptic transmission and relieve neuroprotective effects such as stroke, epilepsy, and neurodegenerative disease symptoms. Tau is mainly transported into the cell by the taurine transporter (TauT; gene sequence Slc6a6, NC_005103.4) on the cell membrane to maintain a high concentration of Tau in the cell. TauT is composed of 621 amino acid residues A protein of about 74 kDa, containing 12 transmembrane helical regions. After acute injury, intr...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/12A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03C07K14/47C12N15/907
Inventor 黄慧玲王辰范维佳莫丽冬徐丽霞仝慧慧齐浩铭
Owner 天津市环湖医院
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