Pair of transcriptional activation subsample effect factor nucleases and coding gene and application thereof

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

Inactive Publication Date: 2012-08-08
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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  • Pair of transcriptional activation subsample effect factor nucleases and coding gene and application thereof
  • Pair of transcriptional activation subsample effect factor nucleases and coding gene and application thereof
  • Pair of transcriptional activation subsample effect factor nucleases and coding gene 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 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

[0064] (12 is also possible, but may be less efficien...

Embodiment 2

[0068] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0069] 1. Acquisition of TALENs identification module (modular)

[0070] (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.

[0071] table 3

[0072]

[0073]

[0074] (2) Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the connection method is:

[0075] ①Take 3 μl of PCR product;

[0076] ② Add 1 μl pEASY-B vector;

[0077] ③25℃, 7min;

[0078] ④Transform DH5a competent cells and spread kanamycin plate;

[0079] 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.

[0080] 2. Identify connections between modules

[0081] Connection strategy: Take the connection of 19 identification modules as an example to ill...

Embodiment 3

[0152] The transfection of embodiment 3 plasmids

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 5. Passage and transfect at the same time.

[0158] 6. Transfect the constructed PRNP-TALEN-L613, PRNP-TALEN-L614, PRNP-TALEN-L...

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Abstract

The invention discloses a pair of transcriptional activation subsample effect factor nucleases and a coding gene and application thereof. The pair of transcriptional activation subsample effect factor nucleases (TALEN) is obtained by merging a pair of DNA identifying proteins with two different source subunits of a Fok1 DNA incision enzyme respectively, and two adjacent locuses on a prion protein gene (PRNP) exon2 of a goat or a sheep can be identified in specificity mode. When the pair of transcriptional activation subsample effect factor nucleases is simultaneously transferred into a host cell, the nucleases can shoot targets of the exon2 locuses of a host cell PRNP gene and enable the target shooting locuses to have genic mutation so as to perform targeted modification on the PRNP gene of the goat or sheep. The nucleases have the advantages of being strong in specificity, high in target shooting efficiency and 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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