A pair of transcription activator-like effector nucleases and coding engines as well as application thereof

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

Inactive Publication Date: 2012-08-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, unlike the specific triplet bases recognized by each zi

Method used

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  • A pair of transcription activator-like effector nucleases and coding engines as well as application thereof
  • A pair of transcription activator-like effector nucleases and coding engines as well as application thereof
  • A pair of transcription activator-like effector nucleases and coding engines as well as 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 PRNP genome sequences from NCBI (sheep Gene ID: 493887, goat PRNP gene sequence is the same as that of sheep)

[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 13-21 (12 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]

[0073] (2) Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the connection method is:

[0074] ①Take 3 μl of PCR product;

[0075] ② Add 1 μl pEASY-B vector;

[0076] ③25℃, 7min;

[0077] ④Transform DH5a competent cells and spread kanamycin plate;

[0078] 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.

[0079] 2. Identify connections between modules

[0080] Connection strategy: Take the connection of 19 identification modules as an example to ill...

Embodiment 3

[0147] The transfection of embodiment 3 plasmids

[0148] 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% CO2 30min in the incubator.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 5. Passage and transfect at the same time.

[0153] 6. Transfect the constructed PRNP-TALEN-L613, PRNP-TALEN-L614, PRNP-TALEN-L61...

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Abstract

The invention discloses a pair of transcription activator-like effector nucleases and coding engines as well as application thereof. The pair of transcription activator-like effector nucleases (TALEN) is obtained by fusing a pair of DNA (Deoxyribonucleic Acid) recognition proteins with two heterogeneous subunits of Fok1 DNA incision enzyme respectively, and can be used for specifically recognizing two adjacent sites on goat or sheep prion protein gene (PRNP) exon 2. The pair of transcription activator-like effector nucleases is simultaneously transferred into host cells, can be used for targeting exon 2 sites of the host cell PRNP genes and can be used for realizing gene mutation at the targeting sites, so that targeted modification of the goat or sheep PRNP genes is realized; and the pair of transcription activator-like effector nucleases has 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] Prion protein diseases, also known as transmissible spongiform encephalopathy, are a group of fatal infectious central nervous system degenerative diseases, mainly including: scrapie in sheep and goats, bovine spongiform encephalopathy in cattle (ie mad cow disease), and Creutzfeldt-Jakob disease (CJD),...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N9/22C12N15/55C12N15/63C12N5/10C12N15/85C12R1/64
Inventor 肖磊庄庆刚
Owner ZHEJIANG UNIV
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