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Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof

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

Active Publication Date: 2012-10-03
上海煦顼技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to modify certain parts or genes that control pigmentation by modifying their expression levels through fusion between different domains from another protein called an enzyme involved in regulating melanosome formation during development. These modifications have potential benefits such as increased stability against heat stress, reduced production rates caused by defective recessive alleles associated with inherited forms of blindness syndrome, altered immune responses due to abnormalities affecting neurotransmitter function, and improved cancer treatment efficacy based upon changes made throughout the body's own tissues.

Problems solved by technology

This patented technical problem addressed in the patents relating to modifying plants' tracheal circuits involves identifying and controllably altering certain parts within their own cell bodies without affecting others through chemical methods like knockout techniques.

Method used

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  • Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof
  • Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof
  • Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Design of TALENs target sequence

[0052] 1. Download the human RPE65 genome sequence from NCBI (NC 000001.10)

[0053] 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;

[0054] Table 1

[0055]

[0056] 3. Design TALENs recognition sequence (target sequence):

[0057] According to the sequence obtained by sequencing, the recognition sequence of TALENs was determined according to the following principles:

[0058] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th)

[0059] (2) The last base is T

[0060] (3) The length of the recognition sequence is between 13-19

[0061] (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)

[0062] The position of the designed target sequence is as follows ...

Embodiment 2

[0065] Example 2 Connection between TALENs recognition modules and construction of recombinant vector

[0066] 1. Acquisition of TALENs identification module (modular)

[0067] (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.

[0068] table 3

[0069]

[0070] (2) Connect the four fragments into the pEASY-B vector (purchased from Beijing Quanshijin Company), the connection method is:

[0071] 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.

[0072] 2. Identify connections between modules

[0073] 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

[0118] Transfection human 293T cell of embodiment 3 plasmids

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 5. Passage and transfect at the same time.

[0124] 6. The constructed RPE65-TALEN-L1, RPE65-TALEN-L2, RPE65-TAL...

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Abstract

The invention discloses a pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof. The pair of transcription activator-like effector nucleases are obtained from fusing a pair of DNA recognition proteins with two heterogenous subunits of Fok1 DNA endonuclease respectively and can recognize specifically two adjacent sites of human RPE65 gene exon1. When the pair of transcription activator-like effector nucleases are transferred into host cells, the RPE65 gene exon1 site of the host cells can be targeted and then genic mutation occurs at the targeted locus, which achieves the target modification of RPE65 gene; in addition, the TALEN has the advantages of high specificity, high targeting efficiency, high accuracy, and on the like.

Description

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Claims

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Application Information

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Owner 上海煦顼技术有限公司
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