Pair of transcriptional activator-like effector nucleases (TALEN) as well as encoding gene and application thereof

A nuclease, a pair of technology, applied in the field of genetic engineering, to achieve the effect of high targeting efficiency, high accuracy and strong specificity

Active Publication Date: 2016-08-10
江苏好润生物产业集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unlike the specific triplet bases recognized by each zinc finger protein, each RVDs on TALEs can only recognize one base

Method used

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  • Pair of transcriptional activator-like effector nucleases (TALEN) as well as encoding gene and application thereof
  • Pair of transcriptional activator-like effector nucleases (TALEN) as well as encoding gene and application thereof
  • Pair of transcriptional activator-like effector nucleases (TALEN) as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The design of embodiment 1 TALENs target sequence

[0036] 1. Download the human Pax6 gene (GENE ID: 5080) from NCBI;

[0037] 2. Design primers and PCR amplify the targeting site fragments on the genome, and sequence them. The PCR primers and sequencing primers are shown in Table 1;

[0038] Table 1

[0039]

[0040] 3. Design TALENs recognition sequence (target sequence):

[0041] According to the sequence obtained by sequencing, the recognition sequence of TALENs was determined according to the following principles:

[0042] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th);

[0043] (2) The last base is T;

[0044] (3) The length of the recognition sequence is between 13-19;

[0045] (4) The length of the spacer sequence (Spacer) between the two recognition sequences is controlled between 14-21 (12, 13 are also possible, but the efficiency may be low).

[0046] The position of the designed target sequence is as follows...

Embodiment 2

[0049] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0050] 1. Acquisition of TALENs identification module (modular)

[0051] (1) Synthesize four recognition modules NI, NG, HD, and NK that recognize bases A, T, C, and G respectively. The sequences are shown in Table 3.

[0052] table 3

[0053]

[0054]

[0055] (2) Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Co., Ltd.), and directly connect the blunt ends. The connection method is as follows:

[0056] ①Take 3 μL of PCR product;

[0057] ② Add 1 μL pEASY-B vector;

[0058] ③25℃, 7min;

[0059] ④Transform DH5a competent cells and spread on agar plates containing kanamycin;

[0060] ⑤Pick clones, extract plasmids in a small amount, digest, sequence and verify, and finally obtain the recognition modules NI, NG, HD, and NK connected to the vector pEASY-B.

[0061] 2. Identify connections between modules

[0062] Connection st...

Embodiment 3293

[0129] Gene knockout in embodiment 3293T cells

[0130] 1. Plasmid transfection

[0131] (1) Add 100 μL Matrigel to each well of a 6-well plate, shake it back and forth to make it cover the bottom of the entire well, and place it in 5% CO 2 30min in the incubator.

[0132] (2) Aspirate the culture medium in the T25 flask where 293T cells were cultured, and once in PBS, add 1mL of 0.25% trypsin, shake back and forth to make it evenly cover the bottom of the flask, and place in 5% CO 2 5min in the incubator.

[0133] (3) After the digestion was completed, 1 mL of 10% DMEM was added to neutralize the trypsin, and the digested cells were transferred to a 15 mL centrifuge tube, and the cells were counted and centrifuged at 1200 rpm for 5 min.

[0134] (4) The cells were resuspended with an appropriate amount of 10% DMEM, and 2 million 293T cells were placed in a 6-well plate on which Matrigel had been laid, and 2 mL of fresh 10% DMEM was added.

[0135] (5) Passage and transfec...

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Abstract

The invention discloses a pair of transcriptional activator-like effector nucleases (TALEN) as well as an encoding gene and application thereof. The pair of TALENs is obtained by fusing a pair of DNA recognition proteins with one Fok1 nuclease monomer respectively and can specifically recognize two adjacent sites on exon 6 of a human Pax6 gene and carry out digestion. When the pair of TALENs are transferred into host cells at the same time, exon 6 sites of Pax6 genes of the host cells can be targeted, and gene mutation is carried out on the targeted sites, so that targeting modification on the human Pax6 gene is realized, and the advantages of strong specificity, high targeting efficiency and high accuracy are realized.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a pair of transcription activator-like effector nucleases and their coding genes and applications. Background technique [0002] The Pax6 gene plays an important role in the development of the animal's central nervous system, eyes, nose, pancreas and pituitary gland. Mutations in the Pax6 gene lead to a variety of diseases and symptoms, such as human aniridia, iris hypoplasia, keratitis, ectopic pupils, nystagmus, neurodevelopmental disorders, optic nerve dysplasia, etc., microphthalmia in mice, and absence of eyes in Drosophila. Pax6 gene is a highly conserved transcription factor located at 11q13-14, and its encoded protein Pax6 consists of DNA binding domain and transcription activation domain from N-terminus to C-terminus. [0003] It has always been the dream of many scientists to modify the genome according to human wishes. Specifically delete or add the sequences we ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N5/071
CPCC07K14/47C12N9/22
Inventor 李文静林喜娟肖磊
Owner 江苏好润生物产业集团股份有限公司
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