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A pair of transcription activator like effector nucleases of L3 and R1 and a coding gene and an application thereof

A technology of transcriptional activation and effectors, applied in the field of genetic engineering

Active Publication Date: 2013-01-02
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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  • A pair of transcription activator like effector nucleases of L3 and R1 and a coding gene and an application thereof
  • A pair of transcription activator like effector nucleases of L3 and R1 and a coding gene and an application thereof
  • A pair of transcription activator like effector nucleases of L3 and R1 and a coding gene and an application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The design of embodiment 1 TALENs target sequence

[0054] 1. Download the human RHCE gene (GENE ID: 6006) and RHD genome sequence (GENE ID: 6007) from NCBI

[0055] 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;

[0056] Table 1

[0057]

[0058] 3. Design TALENs recognition sequence (target sequence):

[0059] According to the sequence obtained by sequencing, the recognition sequence of TALENs was determined according to the following principles:

[0060] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th)

[0061] (2) The last base is T

[0062] (3) The length of the recognition sequence is between 13-19

[0063] (4) The length of the spacer sequence (Spacer) between the two recognition sequences is controlled between 14-21 (12,13 are also available, but the efficiency may be lower)

[0064] The position of the d...

Embodiment 2

[0067] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0068] 1. Acquisition of TALENs identification module (modular)

[0069] (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.

[0070] table 3

[0071]

[0072] (2) Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the connection method is:

[0073] 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.

[0074] 2. Identify connections between modules

[0075] 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

[0120] Transfection human 293T cell of embodiment 3 plasmids

[0121] 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.

[0122] 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.

[0123] 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 5min.

[0124] 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.

[0125] 5. Passage and transfect at the same time.

[0126] 6. Combine the constructed RH-TALEN-L1, RH-TALEN-L2, RH-TALEN--L...

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Abstract

The invention discloses a pair of transcription activator like effector nucleases, a coding gene and an application thereof. The pair of transcription activator like effector nucleases (TALEN) is obtained by respective fusion of a pair of DNA recognins with two heterogenous subunits of a Fok1 DNA endonuclease, and can specifically recognize two adjacent loci on human RHD or RHCE gene exon1. When the pair of transcription activator like effector nucleases is simultaneously transferred to a host cell, targeting of the exon1 loci of the host cell gene can be realized; the targeted loci are subject to gene mutation; therefore, targeting modification of the human RHCE or RHD gene is realized, and advantages of strong specificity, high targeting efficiency, high accuracy and the like are provided.

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] In addition to the ABO blood group, the common blood group system also has the Rh blood group. Rh blood type is divided into Rh+ and Rh-. RH-positive people can receive blood from RH-negative people, but RH-negative people cannot accept blood from RH-positive people, because the antigens in RH-positive blood will stimulate RH-negative people to produce RH antibodies. If RH positive blood is transfused again, it can lead to hemolytic transfusion reaction. According to relevant information, the Rh-positive blood type accounts for about 99.7% of the Han and most ethnic groups in China, so the Rh- blood type is extremely rare in my country, and many patients lose their lives because they do not have suitable blood. Therefore, Rh- blood is also known as...

Claims

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Application Information

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