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Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof

A one-to-one, coding technology, applied in the field of genetic engineering, to achieve the effects of high targeting efficiency, high accuracy and strong specificity

Active Publication Date: 2014-08-06
浙江煦顼技术有限公司
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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 transcription activator-like effector nucleases (TALEN), encoding gene and application thereof
  • Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof
  • Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Design of TALENs target sequence

[0052] 1. Download the human RPE65 genome sequence from NCBI (NC 000001.10)

[0053] 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;

[0054] Table 1

[0055]

[0056] 3. Design TALENs recognition sequence (target sequence):

[0057] According to the sequence obtained by sequencing, the recognition sequence of TALENs was determined according to the following principles:

[0058] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th)

[0059] (2) The last base is T

[0060] (3) The length of the recognition sequence is between 13-19

[0061] (4) The length of the spacer sequence (Spacer) between the two recognition sequences is controlled between 13-21 (12 is also possible, but the efficiency may be lower)

[0062] The position of the designed target sequence is as follows ...

Embodiment 2

[0065] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0066] 1. Acquisition of TALENs identification module (modular)

[0067] (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.

[0068] table 3

[0069]

[0070] (2) Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the connection method is:

[0071] ①Take 3 μl of PCR product; ②Add 1 μl pEASY-B vector; ③25°C, 7min; ④Transform DH5a competent cells, spread kanamycin plate; The recognition modules NI, NG, HD, NK linked into the vector pEASY-B were obtained.

[0072] 2. Identify connections between modules

[0073] Connection strategy: Take the connection of 19 identification modules as an example to illustrate the connection strategy. Since the last half of the module that can recognize the base T is already on the carrier, it only nee...

Embodiment 3

[0118] Transfection human 293T cell of embodiment 3 plasmids

[0119] 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.

[0120] 2. Aspirate the culture medium in the T25 bottle of cultured IPS cells, suck the PBS once, add 1mL of 0.25% trypsin, shake back and forth to make it evenly cover the bottom of the bottle, and place in 5% CO 2 5min in the incubator.

[0121] 3. After digestion, add 1ml 10% DMEM to neutralize the trypsin, transfer the digested cells to a 15ml centrifuge tube, count the cells, and centrifuge at 1200rpm for 5min.

[0122] 4. Resuspend the cells with an appropriate amount of 10% DMEM, take 2 million 293T cells and place them in a 6-well plate that has been covered with Matrigel, and add 2ml of fresh 10% DMEM.

[0123] 5. Passage and transfect at the same time.

[0124] 6. The constructed RPE65-TALEN-L1, RPE65-TALEN-L2, RPE65-TAL...

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Abstract

The invention discloses a pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof. The pair of transcription activator-like effector nucleases are obtained from fusing a pair of DNA recognition proteins with two heterogenous subunits of Fok1 DNA endonuclease respectively and can recognize specifically two adjacent sites of human RPE65 gene exon1. When the pair of transcription activator-like effector nucleases are transferred into host cells, the RPE65 gene exon1 site of the host cells can be targeted and then genic mutation occurs at the targeted locus, which achieves the target modification of RPE65 gene; in addition, the TALEN has the advantages of high specificity, high targeting efficiency, high accuracy, and on the like.

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 human RPE65 gene is called the retinal pigment epithelium gene, which encodes an important protein with a molecular weight of 65kDa in the retinal pigment epithelium, and participates in key steps such as the circulation of retinal and other substances, and the regeneration of visual pigment rhodopsin. If people lack the protein encoded by this gene, the 11-cis-retinal will be missing, and the rod cells will not respond to light stimulation. The mutation of this gene will lead to Leber congenital amaurosis (LCA2) and retinitis pigmentosa. [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 need on the endogenous genome. On the one hand, various animal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N9/22C12N15/55C12N15/63C12N5/10C12N15/85
Inventor 肖磊赵金龙吴昭
Owner 浙江煦顼技术有限公司
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