Open-source method for screening zinc finger proteins targeted combined with target sites of human DYRK1A gene

A zinc finger protein, targeted binding technology, applied in the field of genetic engineering, can solve the problems of limited zinc finger library capacity, inability to triple base screening, inability to achieve screening, etc., and achieve high efficiency, specificity and high affinity.

Inactive Publication Date: 2011-10-05
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The zinc finger library has a limited storage capacity and cannot screen triple bases such as ANN or CNN, and laboratories without these zinc finger libraries cannot achieve screening

Method used

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  • Open-source method for screening zinc finger proteins targeted combined with target sites of human DYRK1A gene
  • Open-source method for screening zinc finger proteins targeted combined with target sites of human DYRK1A gene
  • Open-source method for screening zinc finger proteins targeted combined with target sites of human DYRK1A gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Construction of 3 zinc finger protein libraries:

[0038] 1. ZF1 (zinc finger 1, zinc finger domain 1) random ZFP1 library construction

[0039] 1.1. Construction of random ZFP1 fragments of ZF1

[0040] Using the zinc finger protein expression plasmid pGP-FB-orig BA as the substrate, ZF1-F and ZFR as primers, use pfu polymerase to amplify according to the PCR reaction system shown in Table 1. The cycle conditions are: 94°C for 5min, 94°C for 30s, 55°C for 30s, 72°C for 30s, a total of 28 cycles, and finally 72°C for 10min. Randomize the 21 key bases in ZF1, and then use this PCR reaction product as a substrate, ZFF and ZFR primers, use pfu polymerase according to the table The PCR reaction system shown in 2 was amplified, and the cycle conditions were: 94°C for 5 minutes, 94°C for 30s, 55°C for 30s, 72°C for 30s, a total of 20 cycles, and finally 72°C for 10 minutes to amplify the complete zinc finger protein expression fragment, namely ZF1 random ZFP1 fragment (R...

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Abstract

The invention, which relates to an open-source method for screening zinc finger proteins targeted combined with target sites of human DYRK1A gene, belongs to the field of gene engineering technology. The method provided by the invention comprises the following steps of: (1) construction of three zinc finger protein libraries; (2) construction of reporter vectors and reporter strains; (3) screening of effective zinc finger libraries; (4) connection and assembly of effective zinc finger libraries; (5) screening of three zinc-fingers protein having high specificity and affinity by the bacterial two-hybrid principle. The method provided by the invention has advantages of high specificity and affinity of obtained zinc finger proteins and higher efficiency of assembled zinc-finger nucleases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an open-source method for screening zinc finger proteins that target and bind to the target site of the human DYRK1A gene. Background technique [0002] Zinc finger nuclease technology is a newly developed emerging technology that can be used in the fields of gene function analysis, animal and plant transgenic research, and gene therapy. Zinc finger nuclease (Zinc finger nuclease, ZFN) is an artificial synthetic enzyme combined with zinc finger protein (Zinc finger protein, ZFP) and non-specific nuclease. The DNA sequence of a specific base sequence is bound to this site; the C-terminus is the non-specific nuclease Fok I domain. ZFN can recognize a specific DNA sequence, and two designed complementary ZFN molecules specifically bind to the target DNA in the form of a dimer at the same time, and cut at 5-7 bp in the middle to generate a double-stranded nick...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12N15/70C40B50/06C40B20/04C12R1/19
Inventor 张智英李战伟杨涵江任刚王令张存芳张婷婷王瑞贾杰只雷洁李亚明支旭勃韩新艳辛颖
Owner NORTHWEST A & F UNIV
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