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TALEN and pMD18 vector-based site-directed mutagenesis system and its application

A technology of TALEN-L and site-directed mutagenesis, which is applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, hydrolytic enzymes, etc., can solve the problems of inability to directly obtain 100% edited cells and low efficiency, and achieve simplification of manual labor, The effect of clear genetic background, efficient and precise editing

Inactive Publication Date: 2014-12-17
SHAOXING PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main defect of the current TALEN technology is that the efficiency is relatively low, generally at 5%-20%, and it is impossible to directly obtain 100% edited cells. Only positive monoclonal cells can be selected and amplified into stable strains.

Method used

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  • TALEN and pMD18 vector-based site-directed mutagenesis system and its application
  • TALEN and pMD18 vector-based site-directed mutagenesis system and its application
  • TALEN and pMD18 vector-based site-directed mutagenesis system and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0069] AR Sequence Analysis of Human Androgen Receptor Gene

[0070] The sequence of the human androgen receptor gene AR (NG_009014.2) was downloaded from the NCBI database. Sequence analysis shows that the gene includes 8 exons and 7 introns, such as figure 2 shown. According to the general principle of gene knockout, the second and third exons of the gene are ideal knockout target sites, and the second exon is used as the knockout target sequence in this embodiment.

[0071] sequence design

[0072] According to the sequence characteristics of the human androgen receptor gene AR, we selected the sequence on the second exon as the target sequence of TALEN. The nucleotide sequence of the second exon of the human androgen receptor gene AR is as follows: figure 2 As shown, according to the TALEN principle, the nucleotide sequences SEQ ID NO.1 and SEQ ID NO.2 with the structure T (N11-18) are used as the target sites recognized by TALEN, where N is A, G, T and C any base. ...

Embodiment 2

[0084] The difference between Example 2 and Example 1 is: ① The purpose of Example 2 is to construct five human androgen receptor AR point mutants, while the purpose of Example 1 is to knock out AR and make it inactive; ② Based on the difference between Example 2 and Example 1, the recognition sites of TALEN-L and TALEN-R are downstream of the 8th exon of the human androgen receptor AR gene, such as figure 2 shown; ③ based on the point mutation sites in Example 2 are located in the 8th exon of the AR gene, so they can share the right arm of homologous recombination, such as Figure 11 As shown, it was amplified by primers SEQ ID NO.18 and SEQ ID NO.19; ④The five kinds of AR point mutants were all artificially designed and synthesized primers to bring in the mutation site, and the mutation site was all downstream primers (such as SEQ ID NO. 10, 12, 14, 15, 17), the product is as follows Figure 6-10 shown. Moreover, there is a puromycin resistance gene between the upstream a...

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Abstract

The invention relates to a TALEN and pMD18 vector-based site-directed mutagenesis system and its application. The system is composed of nuclease TALEN and a pMD18 vector, the nuclease TALEN is a transcription activator-like effect factor shearing a target gene target sequence, and the TALEN is composed of nuclease TALEN-L identifying the 5'-terminal of the target sequence and nuclease TALEN-R identifying the 3'-terminal of the target sequence; and the pMD18 vector is a pMD18 homologous recombinant template plasmid inserted into human AR site-directed mutagenesis gene, and the pMD18 vector is composed of corresponding upstream and downstream sequence of the target gene, and a puromycin screening marker. The system allows genome to be efficiently and accurately edited by using the homologous sequence provided by the Pmd18 vector during the cut-out of a double strand DNA molecule by the TALEN; and puromycin resistant gene is carried out when the introduced homologous sequence is adopted to edit the DNA sequence, so manual labor in the screening process is substantially reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human AR gene site-directed mutation system based on TALEN and pMD18 vectors and the application of the genome-level site-directed mutation system. 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 retro...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/85
CPCC12N9/22C12N15/85
Inventor 宋春娇茹国美郎娟郭艳张林
Owner SHAOXING PEOPLES HOSPITAL
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