Transcription activator-like effector nucleases, and encoding genes 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

Inactive Publication Date: 2014-09-17
ZHEJIANG UNIV
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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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  • Transcription activator-like effector nucleases, and encoding genes and application thereof
  • Transcription activator-like effector nucleases, and encoding genes and application thereof
  • Transcription activator-like effector nucleases, and encoding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 Design of TALENs target sequence

[0054] 1. Download goat and sheep BLG genome sequences from NCBI (GenBank number: Z33881.1 for goats, X12817.1 for sheep), and select exon2 as the target;

[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:

[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 14-19

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

...

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;

[0074] ② Add 1 μl pEASY-B vector;

[0075] ③25℃, 7min;

[0076] ④Transform DH5a competent cells and spread kanamycin plate;

[0077] ⑤Pick clones, extract plasmids in a small amount, digest, and sequence, and finally obtain the recognition modules NI, NG, HD, and NK connected to the vector pEASY-B.

[0078] 2. Identify connections between modules

[0079] Connection strategy: Take the connection of 19 identification modules as an example to illustrate the ...

Embodiment 3

[0153] Example 3 Transfection of plasmids

[0154] 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.

[0155] 2. Aspirate the culture medium in the T25 bottle for culturing IPS cells, and once in PBS, 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.

[0156] 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.

[0157] 4. Resuspend the cells with an appropriate amount of 4*Dox ES0, take 2 million IPS cells and place them in a 6-well plate that has been covered with Matrigel, and add 2ml of fresh 4*Dox ES0.

[0158] 5. Passage and transfect at the same time.

[0159] 6. Transfect the constructed BLG-TALEN-L1, BLG-TALEN-L2, BLG-TALEN-L3, BLG-TALEN-R...

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Abstract

The invention discloses a pair of transcription activator-like effector nucleases, and encoding genes and application thereof. The pair of transcription activator-like effector nucleases (TALEN) is obtained by fusing a pair of deoxyribonucleic acid (DNA) recognition proteins and two heterologous subunits of a Flavobacterium okeanokoites 1(Fok 1) DNA incision enzyme and can recognize two adjacent loci on goat or sheep beta lactoglobulin gene (BLG) exon2 specifically. When the transcription activator-like effector nucleases are transferred to a host cell simultaneously, the exon2 loci of the BLG gene of the host cell can be targeted by the transcription activator-like effector nucleases, and the targeted loci are subjected to genetic mutation, so that the targeted modification of the goat or sheet BLG gene is realized, and the transcription activator-like effector nucleases have the advantages of high specificity, high targeting efficiency, high accuracy and 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] 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 models can be constructed for basic biological research and disease mechanism research. On the other hand, animal reactors can be produced to produce what we need cheaply. Biological components that are difficult to obtain from other sources. [0003] Among them, the use of large animal mammary glands to produce medicinal or health-care proteins not only has huge economic value, but also has social value such as greatly reducing overall medical costs. The principle of the large animal mammary gland is to knock the target protein into the high-efficie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N9/22C12N15/55C12N15/63C12N5/10
Inventor 肖磊赵金龙
Owner ZHEJIANG UNIV
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