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ZMIZ1-mediated targeting knockout transcription activator-like effector nuclease, preparation method and applications

A nuclease and nucleotide technology, applied in the fields of biotechnology and genetic engineering, can solve the problems of low efficiency and wide application

Inactive Publication Date: 2013-12-04
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] People have not found a simple and efficient method for targeted modification of the genome. Traditional gene targeting relies on the gene targeting technology of random exchange of homologous chromosomes that occur naturally in cells, and the efficiency is very low, usually only 10 -6 -10 -8 , this targeting method has only been widely used in mice, and is not widely used in other model animals and large mammals due to its low efficiency
[0005] Therefore, there is currently no mature technology for targeting the human ZMIZ1 gene in this field, so there is an urgent need in this field to carry out research on efficient targeting of the human ZMIZ1 gene

Method used

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  • ZMIZ1-mediated targeting knockout transcription activator-like effector nuclease, preparation method and applications

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Experimental program
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Effect test

Embodiment 1

[0113] Design of TALENs module sequence

[0114] 1. Download the genome sequence of human ZMIZ1 (GeneID: 57178) from NCBI as the target.

[0115] 2. Design primers and perform PCR to amplify the target site fragments on the genome and sequence them.

[0116] The sequences of PCR primers and sequencing primers are listed in Table 1.

[0117] Table 1

[0118]

[0119] 3. Design TALENs recognition sequence: Determine the TALENs recognition sequence according to the sequencing sequence and the following principles:

[0120] The base at position 0 is T (referring to the base preceding the first position adjacent to the recognition sequence);

[0121] The last base is T;

[0122] The length of the recognition sequence is between 14-19;

[0123] The length of the spacer sequence between the two recognition sequences is controlled between 12-21, preferably between 14-21,

[0124] The position of the designed target sequence is as follows figure 1 , the sequence of the identi...

Embodiment 2

[0128] Connection between TALENs recognition modules and construction of recombinant vector

[0129] 1. Obtaining the identification module

[0130] The sequences of four recognition modules NI, NG, HD, and NK (SEQ ID NO: 14-17) for synthesizing recognition bases A, T, C, and G are shown in Table 3.

[0131] table 3

[0132]

[0133]

[0134] Ligate the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the ligation method is as follows: take 3 μl of PCR primers; add 1 μl of pEASY-B vector; 25°C, 7min; transfect DH5α competent cells, Coat the kanamycin plate; pick clones, extract plasmids in a small amount, digest and sequence; finally obtain the recognition templates NI, NG, HD and NK connected to the vector pEASY-B.

[0135] 2. Identify connections between modules

[0136] Connection strategy: Take the connection of 19 recognition modules as an example to illustrate the connection strategy, because the last half module that can recog...

Embodiment 3

[0203] The transfection of embodiment 3 plasmids

[0204] 1. Add 300 μl of gelatin to each space of the 6-well plate, shake it back and forth, so that the gelatin covers the bottom of the entire well, and place it in a 5% CO2 incubator for 10 minutes after spreading.

[0205] 2. Aspirate the culture medium in the T25 bottle of cultured 293T cells, wash it once with PBS, add 1ml of 0.25% trypsin, shake back and forth to make it evenly cover the bottom of the bottle, and place it in a 5% CO2 incubator for 5 minutes.

[0206] 3. After digestion, add 1ml 10% DMEM to neutralize trypsin, transfer the digested cells to a 15ml centrifuge tube, count the cells, and centrifuge at 1200rpm for 5 minutes.

[0207] 4. Resuspend the cells with an appropriate amount of 10% DMEM, take 2 million 293T cells and place them in a 6-well plate covered with gelatin, and add 2ml of fresh 10% DMEM.

[0208] 5. Transfect when the density of 293T cells reaches 80%-90%, no need to change the medium.

[...

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Abstract

The invention relates to ZMIZ1-mediated targeting knockout transcription activator-like effector nuclease, a preparation method and applications, and concretely relates to an artificially synthetic method for a transcription activator-like effector nuclease (TALEN) gene which can target human endogenous ZMIZ1 gene efficiently, and a directed targeting method of recombinant plasmids containing the TALEN gene. The transcription activator-like effector nuclease contains a pair of transcription activator-like effector (TALEs) proteins and catalytic subunits, of DNA endonucleases, which are fused with the proteins respectively, and has functions of identifying and cutting two adjacent loca of the human ZMIZ1 gene respectively. Based on the design of the amino acid sequence of TALEs, the nucleotide sequence coding the TALEN is synthetized and the carrier containing the nucleotide sequence is constructed. Through transfection of cells by utilization of TALENs plasmids, the targeting efficiency of cells can be raised greatly.

Description

technical field [0001] The present invention relates to the fields of biotechnology and genetic engineering, in particular, the present invention relates to a transcriptional activator-like effector nuclease mediating ZMIZ1 targeted knockout and its preparation method and application. Background technique [0002] ZMIZ1 (GeneID: 57178), also known as MIZ, RAI17, ZIMP10, hZIMP10, is a gene located on human chromosome 10 (10q22.3). PIAS (protein inhibitor of activated STAT) encoded by this gene has regulatory effects on various transcription factors (such as androgen receptor (AR), Smad3 / 4, and P53) and ubiquitination process. Studies have shown that the gene locus on chromosome 10 and the tyrosine protein kinase (ABL1) locus on chromosome 9 are associated with acute lymphoblastic leukemia after chromosomal translocation. [0003] TGF-β plays an important role in cell proliferation, differentiation and apoptosis. Smad-like proteins are components of TGF-β type I receptors, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N9/22C12N15/55C12N15/63C12N5/10A61K48/00A61K38/16A61K38/46C12N15/85C12N15/12
Inventor 肖磊庄庆刚
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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