Method for targetedly activating gene transcription on basis of natronobacterium gregoryi Argonaute

A technology of gene editing and endogenous genes, applied in genetic engineering, chemical instruments and methods, biochemical equipment and methods, etc.

Active Publication Date: 2017-10-20
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention solves the recent problems of NgAGO application, and provides s

Method used

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  • Method for targetedly activating gene transcription on basis of natronobacterium gregoryi Argonaute
  • Method for targetedly activating gene transcription on basis of natronobacterium gregoryi Argonaute
  • Method for targetedly activating gene transcription on basis of natronobacterium gregoryi Argonaute

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] In the following examples, taking the Nanog gene as an example, the research on the activation of intracellular genes by using NgC (fused VP64 to the C-terminal of NgAGO) and NgN (fused VP64 to the N-terminal of NgAGO) was carried out. Example 1 Construction of NgAGO-VP64 fusion protein

[0041] 1. Fusion expression of VP64 and NgAGO

[0042] In order to prevent the influence of the relative position of the protein on the protein activity, VP64 was fused to the C-terminal (NgC) and N-terminal (NgN) of NgAGO respectively, and the protein sequences were shown in SEQ ID NO.1 and SEQ ID NO.2 respectively (gene structure Schematic such as figure 1 As shown, the sequence composition is as follows figure 2 and image 3 shown).

[0043] 2. After the protein sequence is optimized for humanized codons, it is cloned into the mammalian expression vector pCDNA3.1 respectively. After the plasmid was verified to be correct by sequencing, the plasmid was extracted and used for subs...

Embodiment 2

[0044] Example 2 Guided DNA Synthesis and Phosphorylation

[0045] 1. Design the guide DNA sequence, as shown in Table 1:

[0046] Table 1

[0047]

[0048] The guide DNA (gDNA) is 24bp in length and dissolved to 100μM with 1XT4PNK buffer (NEB) after synthesis. Phosphorylate with 5 units of T4PNK per 1nmolgDNA, and incubate overnight at 37°C. Then store at -20°C for later use.

Embodiment 3

[0049] Example 3 NgAGO genome editing tool composed of NgAGO-VP64 fusion protein and guide DNA and its application

[0050] 1. An NgAGO genome editing tool based on Halophilus grisii Argonaute targeted activation gene transcription, comprising the NgAGO-VP64 fusion protein described in Example 1 and the guide DNA described in Example 2.

[0051] 2. NgAGO genome editing tool activates gene transcription experiment

[0052] (1) Cell culture and transfection

[0053] The day before transfection, the cells were subcultured so that the cells were in the exponential growth phase at the time of transfection. Taking Hela cells as an example, 100,000 cells can be plated in each well of a 12-well plate. One hour before transfection, the cell culture medium was changed to serum-free medium and incubated for 1 hour. Transfection was performed using Lipofactamine2000 transfection reagent.

[0054] Taking the amount per well in a 12-well plate as an example, dilute 1 μg fusion protein p...

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Abstract

The invention discloses a NgAGO-VP64 fusion protein, a guided DNA (gDNA) and a method for targetedly activating gene transcription on the basis of natronobacterium gregoryi Argonaute (NgAGO). The sequence of the NgAGO-VP64 fusion protein is shown as SEQ ID NO.1 or SEQ ID NO.2. The guided DNA is any one or more of mixtures among sequence segment shown as SEQ ID NO.3-12. By utilizing the NgAGO-VP64 fusion protein, the guided DNA and transfection reagent to cotransfect cells, endogenous gene transcription can be targetedly activated. The invention proves that NgAGO has the capability of combining with the guided DNA and identifying target genome DNA. The invention discovers for the first time that the fusion protein constructed from NgAGO and VP64 has the capability of activating endogenous gene in cells. The invention solves the problem that people doubt the application of NgAGO recently, and provides support for the development of target genome editing tools and other tools.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a method for activating gene transcription based on Halophilus grisii Argonaute (NgAGO). Background technique [0002] Gene editing technology refers to the ability of humans to "edit" target genes to achieve knockout and addition of specific DNA fragments. It has shown great potential in basic research, gene therapy and genetic improvement. Therefore, genome editing tools have great theoretical and clinical application value. [0003] At present, new treatments for AIDS and tumors based on ZFN and CAS9 gene editing tools have entered clinical trials. Moreover, the results of initial clinical trials have proved its effectiveness. Compared with ZFN genome editing tools based on protein and DNA recognition, CAS9, as a base pairing recognition mechanism based on RNA and DNA, has the advantages of easy operation and large-scale construction. However, since CAS9 requi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/63C12N15/113C12N9/22
CPCC07K14/215C07K2319/035C12N9/22C12N15/113C12N2310/10
Inventor 徐建勇
Owner SHENZHEN UNIV
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