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Zinc finger nuclease and application thereof

A technology of zinc finger nuclease and zinc finger protein, which is applied in the field of genetic engineering, can solve the problems of T cell incompetence and affect the immune activity of T lymphocytes, and achieve high cutting efficiency, feasible gene therapy, and high targeting accuracy

Pending Publication Date: 2019-11-22
浙江煦顼技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this autologous T cell immunotherapy has limitations
For example, adrenal glucocorticoid receptors are widely distributed on the surface of various cells. Adrenal glucocorticoid receptors on the surface of different cells bind to ligands and trigger different cellular responses. Adrenal glucocorticoids bind to receptors on the surface of T lymphocytes Can cause T cell anergy and affect the immune activity of T lymphocytes

Method used

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  • Zinc finger nuclease and application thereof
  • Zinc finger nuclease and application thereof
  • Zinc finger nuclease and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0146] Design of TRAC-specific ZFNs. Construct TRAC-specific ZFNs to enable the specific introduction of mutation sites on the TRAC gene (such as figure 1 shown). ZFN is composed of two parts, the zinc finger DNA binding domain responsible for the specific recognition sequence and the DNA cleavage domain for non-specific restriction endonuclease cleavage. The zinc finger binding domain generally includes 3-5 independent zinc finger (Zinc finger, ZF) repeat structures, and each zinc finger structure can recognize 3 bases. The most widely used DNA cleavage domain in ZFNs comes from the restriction enzyme FokI. Since the cleavage domain binds weakly to the DNA strand, the DNA cleavage domain must function as a dimer. When constructing a zinc finger nuclease, two ZFNs should be designed for the adjacent regions on each strand of DNA, that is, the ZFN left arm and the ZFN right arm, so that the DNA cleavage domain can be located at the same position on the double strand to achie...

Embodiment 2

[0154] ZFN activity test in vitro. The ZFN-left arm plasmid vector and the ZFN-right arm plasmid vector were transfected into Hela cells using Fugene transfection reagent, respectively. 24 hours after transfection, HeLa cells were treated with 1 μg / ml puromycin for 48 hours to obtain ZFN-enriched cells. HeLa cells were then harvested, and the cleaved DNA fragment containing the ZFN was amplified by PCR using TRAC gene-specific primers and the HeLa cell genome as a template. DNA fragments are sequenced using the forward primer. The cleavage domain of the ZFN comprises a wild-type FokI cleavage domain (SEQ ID NO.8 as shown in Table 2) or an engineered FokI cleavage domain (SEQ ID NO.17 or 18 as shown in Table 2) .

[0155] Cloning of DNA fragments into vectors. The DNA fragments of about 30 monoclonal cells were sequenced to determine whether the DNA fragments included mutations. Double sequencing signals were found at the TRAC site, indicating that the ZFN of the present i...

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Abstract

The invention discloses a zinc finger nuclease and application thereof, and belongs to the technical field of genetic engineering. The related the zinc finger nuclease (ZFN) comprises a first zinc finger protein (ZFP) bonded with a first target site in a T cell receptor (TRAC) gene and a second ZFP bonded with a second target site in the TRAC gene, can perform targeted identification and cutting on specific target DNA, and has high cutting efficiency, so that the introduction of zinc finger nucleotidase cutting genome DNA into the cells can effectively improve the recombination rate.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to a zinc finger nuclease (ZFN) and its application. Background technique [0002] Genome editing technologies (GETs) can precisely target and modify specific sites in the genome of organisms and artificially modify the genetic information of organisms. Zinc finger nucleases (zinc finger nucleases, ZFN), transcription activator-like effector nucleases (transcription activator-like effector nucleases, TALEN) and clustered regularly interspaced short palindromic repeats and related systems (clustered regularly interspaced short palindromic The new genome editing technology represented by repeats / Cas endonucleases (CRISPR / Cas) has set off an upsurge in life science research and has been widely used in various fields such as biology, medicine, and agriculture. Different from the traditional transgenic modification technology that produces random site integra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N5/10
CPCC12N9/22C12N5/0636C12N5/0646C12N2510/00
Inventor 肖磊吴昭毛丽刘婧睿
Owner 浙江煦顼技术有限公司
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