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A pair of transcription activator-like effector nucleases and coding sequence and application thereof

A nucleotide sequence, a pair of technology, applied in the field of genetic engineering, can solve the problem of outbred mouse model construction that has not been reported

Active Publication Date: 2016-03-30
SHENZHEN HUADA GENE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, all current colitis research focuses on inbred mice, and the establishment of outbred mouse models has not been reported

Method used

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  • A pair of transcription activator-like effector nucleases and coding sequence and application thereof
  • A pair of transcription activator-like effector nucleases and coding sequence and application thereof
  • A pair of transcription activator-like effector nucleases and coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1, Sequence Analysis of Mouse Ace2 Gene and Design of TALEN Targeting Site

[0038] The NCBI database shows that the mouse Ace2 gene (gene ID: 70008) has a full length of 49,077bp, which encodes an mRNA consisting of 19 exons, and further encodes a protein consisting of 805 amino acid residues. The gene structure is as follows: figure 1 shown.

[0039] According to the structural characteristics of the mouse Ace2 gene, in this embodiment, the second exon is used as the TALEN targeting site, and the recognition site of the TALEN on the mouse Ace2 gene is selected as 5'- TCACCGAG GAAAATGCCAA GACATTTTTAAACAAC TTTAATCAGGAAGCTGA -3', where the underlined parts are TALEN-L and TALEN-R recognition sites, namely SEQ ID NO.3 and SEQ ID NO.4.

[0040] According to the principle of the variable module NI to recognize A base, NG to recognize T base, NN to recognize G base, and HD to recognize C base, according to the method of GoldenGate, design a polypeptide that spe...

Embodiment 2

[0043] Embodiment 2, the preparation of Ace2 gene knockout mouse

[0044] The mRNA prepared in Example 1 was mixed together at a final concentration of 50 ng / uL each of TALEN-L and TALEN-R, and the mixture was injected into the cytoplasm of fertilized eggs of Kunming mice, with an injection volume of 1 pL to 50 pL. Afterwards, the surviving fertilized eggs were transplanted back into the wombs of surrogate mice. After a gestation period of about 21 days, a total of 49 mice were born. Take the tail tip of the mouse, and use the phenol-chloroform method to extract DNA. Whether mouse targeting is successful is determined by RFLP-DraI, specifically:

[0045] The following primers were used with the sequence:

[0046] ACE2-F:5'-CTTCTCAGTGCCCAACCCA-3'

[0047] ACE2-R:5'-GGATCAGAGCTACAGAGGCAGT-3'

[0048] Using mouse DNA as a template, PCR amplification was performed to obtain a 432bp amplified product, which included the sequence recognized by TALEN-L and TALEN-R, and the DraI ...

Embodiment 3

[0051] Embodiment 3, establishment of colitis mouse model

[0052] From the Ace2 gene knockout mice prepared in Example 2, select male mice with biallelic knockouts and divide them into two groups at random, with 6 mice in each group: feeding one group of mice with common drinking water, Denoted as "Ace2 knockout, control"; another group was fed with drinking water added with 5% dextran sodium sulfate (DSS), denoted as "Ace2 knockout, DSS". At the same time, Kunming male mice of the same age as the knockout mice were selected and randomly divided into two groups, with 6 mice in each group: one group of mice was fed with ordinary drinking water, which was recorded as "wild type, control"; Another group was fed with drinking water added with 5% dextran sodium sulfate (DSS), which was denoted as "wild type, DSS". During the feeding process, the mice developed diarrhea. Moreover, with the prolongation of feeding time, the degree of diarrhea also intensified, and even blood in th...

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Abstract

The invention discloses a transcription activator-like effector nuclease mediating targeted knockout of a colitis related gene Ace2, a coding sequence and an application of the nuclease. The transcription activator-like effector nuclease disclosed by the invention comprises a pair of transcription activator-like effector proteins and a monomer or a catalytic subunit of a DNA incision enzyme, wherein the monomer or the catalytic subunit are separately fused with the proteins. The transcription activator-like effector proteins have the functions of separately identifying and cutting two adjacent loca of an Ace2 gene of a mouse to efficiently and specifically realize targeted knockout of the Ace2 gene so as to quickly obtain a colitis model of the mouse, thereby providing a good genetic resource for research on colitis.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a pair of transcription activator-like effector nucleases and their coding sequences and applications. Background technique [0002] With the advent of the post-genome era, reverse genetics has become an important means for people to study specific gene functions. The establishment of animal disease models is an indispensable tool for people to study the pathogenesis of specific diseases, screen drug targets and evaluate drug properties. Among them, the mouse model has the advantages of small size, convenient feeding and management, easy control, fast production and reproduction, and low cost of use, and has been favored by researchers. At present, more than 1,000 inbred and outbred strains have been bred through long-term artificial breeding and selective breeding of mice. Among so many mouse strains, due to their high genetic consistency, the data obtained from in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N9/22C12N15/62C12N15/55C12N15/89C12N5/10
Inventor 刘楚新刘欢肖丽萍李飞达张兴举王俊
Owner SHENZHEN HUADA GENE INST
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