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Recombinant transcription activator like effector, transcription activator like effector nuclease, as well as coding gene and application thereof

A transcriptional activation and effector technology, applied in the field of genetic engineering, to achieve high targeting efficiency, high accuracy, and strong specificity

Active Publication Date: 2012-10-03
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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  • Recombinant transcription activator like effector, transcription activator like effector nuclease, as well as coding gene and application thereof
  • Recombinant transcription activator like effector, transcription activator like effector nuclease, as well as coding gene and application thereof
  • Recombinant transcription activator like effector, transcription activator like effector nuclease, as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Design of TALENs target sequence

[0040] 1. Download the human insulin genome sequence from NCBI (NC 000011.9)

[0041] 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;

[0042] Table 1

[0043]

[0044] 3. Design TALENs recognition sequence (target sequence):

[0045] According to the sequence obtained by sequencing, the recognition sequence of TALENs was determined according to the following principles:

[0046] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th)

[0047] (2) The last base is T

[0048] (3) The length of the recognition sequence is between 13-19

[0049] (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)

[0050] The position of the designed target sequence is as follow...

Embodiment 2

[0054] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0055] 1. Acquisition of TALENs identification module (modular)

[0056] (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.

[0057] table 3

[0058]

[0059] (2) Ligate the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the ligation method is as follows: ① Take 3 μl of PCR product; ② Add 1 μl of pEASY-B vector; ③ 25°C, 7min; ④ Transform DH5a sensory State cells, coated with kanamycin plate; ⑤Pick clones, extract plasmids in a small amount, digest, sequence, and finally obtain the recognition modules NI, NG, HD, and NK connected to the vector pEASY-B.

[0060] 2. Identify connections between modules

[0061] Connection strategy: Take the connection of 19 identification modules as an example to illustrate the connection strategy. Since the...

Embodiment 3

[0105] Example 3 Transfection of plasmids into human 293T cells

[0106] 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.

[0107] 2. Aspirate the culture medium in the T25 bottle of cultured IPS cells, suck the PBS once, add 1mL 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.

[0108] 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.

[0109] 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.

[0110] 5. Passage and transfect at the same time.

[0111] 6. Transfect cells with the constructed insulin-TALEN-L1, in...

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Abstract

The invention relates to the field of genetic engineering and discloses a pair of polypeptides and encoding genes thereof, with sequences shown as SEQ ID NO. 1 to 4. The pair of polypeptides is used for establishing a pair of a recombinant transcription activator like effector (TALE) and a transcription activator like effector nuclease (TALEN); the TALEN is a fusion protein formed by fusing a pair of DNA recognizing proteins with a DNA severing protein respectively; and the TALEN can perform targeted severing on a target site of a human insulin gene so as to achieve targeted modification on the human insulin gene, with the characteristics of strong specificity, high efficiency and high accuracy.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a pair of polypeptides related to insulin gene recognition, a pair of recombinant transcriptional activator-like effectors, a pair of transcriptional activator-like effector nucleases, coding genes and applications thereof. Background technique [0002] The human insulin gene is the gene encoding human insulin. Insulin is a protein hormone secreted by pancreatic β cells stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, and glucagon. Insulin is the only hormone that lowers blood sugar in the body, and at the same time promotes the synthesis of glycogen, fat, and protein. Damage to pancreatic β cells and abnormal insulin secretion can cause diabetes. , protein, fat, water and electrolytes and a series of metabolic disorder syndromes, clinically characterized by hyperglycemia, polyuria, polydipsia, polyphagia, weight loss and other manif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N9/22C12N15/55C12N15/63C12N5/10C12N15/85
Inventor 肖磊赵金龙吴昭
Owner 浙江煦顼技术有限公司
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