Transcription activator subsample effector nuclease, and coding gene and application thereof

A transcriptional activation and effector technology, applied in the field of genetic engineering, can solve the problems of unexplored therapeutic effect and difficulty in finding TALEN targeting sequences

Inactive Publication Date: 2015-02-18
SHAOXING PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, the problems related to human miRNAs gene knockout mainly include: 1. The coding sequence of miRNAs is only about 300 nucleotides, and it is difficult to find efficient TALEN targeting sequences; 2. The copy number of miRNAs may be more than one, and members of the same family may also be Multiple, so effective double knockout and multiple knockout are the key issues; 3. miRNAs play a synergistic role, and the therapeutic effect of knocking out a single miRNAs gene remains to be investigated

Method used

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  • Transcription activator subsample effector nuclease, and coding gene and application thereof
  • Transcription activator subsample effector nuclease, and coding gene and application thereof
  • Transcription activator subsample effector nuclease, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Design of TALENs target sequences

[0049] 1. Download human miRNAs: miR-133b, miR-141 and miR-488 gene DNA sequences from miRBase;

[0050] 2. Design TALENs recognition sequence (target sequence):

[0051] According to the sequences obtained from the miRBase database, the recognition sequences of TALENs were determined according to the following principles:

[0052] (1) The 0th base is T (the base before the first in the recognition sequence is the 0th)

[0053] (2) The last base can be AGCT, preferably T

[0054] (3) The length of the recognition sequence is between 13-19

[0055] (4) The length of the spacer sequence between the two recognition sequences is controlled between 14-21 (12,13 are also available, but the efficiency may be lower)

[0056] The position of the designed target sequence is as follows figure 1 The specific sequence is shown in Table 1.

[0057] Table 1

[0058]

Embodiment 2

[0060] Connection between TALENs recognition modules and construction of recombinant vector

[0061] 1. TALENs identification module

[0062] The four recognition modules NI, NG, HD, and NK that respectively recognize bases A, T, C, and G are shown in Table 2 for their sequences.

[0063] Table 2

[0064]

[0065] 2. Identify connections between modules

[0066] Connection strategy: Take the connection of 19 identification modules as an example to illustrate the connection strategy. Since the last module that can recognize the base T is already on the carrier, it only needs to connect 18 modules, and divide the 18 modules into 9 groups in pairs.

[0067] 3. Construction and screening of recombinant vectors

[0068] (1) Construct the left and right arm expression vectors of the nuclease TALEN according to the kit instructions provided by Shanghai Steincell Biotechnology Co., Ltd. Add the reagents corresponding to each module to the above 9 groups in sequence according ...

Embodiment 3

[0071] Human RWPE-1 cells transfected with plasmid

[0072] 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.

[0073] 2. Aspirate the medium in the T25 flask for culturing RWPE-1 cells, aspirate PBS once, add 1mL of 0.25% trypsin, shake back and forth to make it evenly cover the bottom of the flask, and place in 5% CO 2 5min in the incubator.

[0074] 3. Add 1 mL of 10% DMEM to neutralize the trypsin after the digestion is complete, transfer the digested cells to a 15 mL centrifuge tube, count the cells, and centrifuge at 1200 rpm for 5 min.

[0075] 4. Resuspend the cells with an appropriate amount of 10% DMEM, take 2 million RWPE-1 cells and place them in a 6-well plate that has been covered with Matrigel, and add 2 mL of fresh 10% DMEM.

[0076] 5. Subculture and transfect at the same time.

[0077] 6. The constructed miR-133b-TALEN-L and miR-133...

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Abstract

The invention relates to a transcriptional activator subsample effector nuclease, and coding gene and application thereof. The transcriptional activator subsample effector nuclease comprises three pairs of short peptides, wherein the three pairs of short peptides are respectively a first pair of short peptides, a second pair of short peptides and a third pair of short peptides; the first pair of short chain peptides identifies DNA sequences of SEQIDNO.1 and SEQIDNO.2; the second pair of short peptides identifies DNA sequences of SEQIDNO.3 and SEQIDNO.4; the third pair of short peptides identifies DNA sequences of SEQIDNO.5 and SEQIDNO.6. The transcriptional activator subsample effector obtained by establishing the three pairs of short peptides can respectively and specifically identify two adjacent nucleotides near gene seed sequences of human miRNAs: miR-133b, miR-141 and miR-488, and by utilizing the transcriptional activator subsample effector nuclease obtained by establishing the three pairs of transcriptional activator subsample effectors, accurate and efficient targeting can be performed on the genes of human miRNAs: miR-133b, miR-141 and miR-488.

Description

technical field [0001] The invention relates to the field of genetic engineering, specifically transcription activator-like effector nuclease and its coding gene and application, especially for the knockout of human miRNAs gene. Background technique [0002] Genome targeted modification is one of the hotspots in life science research in recent years, especially in gene therapy of human diseases, genome targeted modification has broad application prospects. Change the genetic composition of genes through operations such as transgenic or gene knockout, and obtain genetic engineering tools that meet various needs. The more commonly used traditional transgenic methods are plasmid stable transfection method, retrovirus carrying method and homologous recombination method. However, the insertion site of plasmid transfection stable screening is random and requires antibiotics to maintain, which may easily lead to abnormal cell phenotypes; while the efficiency of retrovirus carrying...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/85
CPCC12N9/22C12N15/113C12N15/85C12N2310/141
Inventor 宋春娇茹国美张伟光杨万雷
Owner SHAOXING PEOPLES HOSPITAL
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