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A drug-induced gene transcription activation method

A gene transcription, drug technology, applied in the field of molecular biology

Active Publication Date: 2020-12-01
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been some successful reports on the use of TALE to achieve drug-induced transcriptional activation, but there are certain limitations in application.

Method used

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  • A drug-induced gene transcription activation method
  • A drug-induced gene transcription activation method
  • A drug-induced gene transcription activation method

Examples

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Embodiment 1

[0049] In this example, a TAL targeting the 14-bp sequence of the promoter region of the human endogenous SOX2 gene is used as a representative to construct a drug-induced TALE system. #35388). One or two serial ERs are fused to the N-terminus of TALE respectively T2 , and then multiple transcriptional activation domains (ADs), including VP64(V), P65(P), Rta(R) and HSF1(H), were fused to the C-terminus of Sox2TALE in different combinations. Both V and H were amplified from plasmids (from Feng Zhang's laboratory, Addgene plasmids#353885and#61426 19) as templates, while P and R were synthesized by the company (Genewiz), and the sequences were referenced from Chavez A.et.Al. (Chavez, A. et al. Highly efficient Cas9-mediatedtranscriptional programming. Nature methods12, 326-328(2015).). TALE-SunTag system-related plasmids are fused to ER with 10 copies of the GCN4 fragment T2 -Sox2TALE or 2ER T2 -Sox2TALE or the C-terminus of 3xNLS-Sox2TALE. In addition, we cloned a series of...

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Abstract

The invention relates to the field of molecular biology, in particular to a drug-induced gene transcription activation method. By transfecting (ERT2)n-TALE-ADs system or co-transfection of HIT TALE-SunTag system into the cytoplasm, 4-OHT and / or TAM and / or their derivatives are utilized in specific time to perform medical induction to achieve transcriptional activation of specific genes. The (ERT2)n-TALE-ADs system is expressible (ERT2)n-TALE-ADs fusion protein vector. The HIT TALE-SunTag system is a vector combination which can express one or more of fusion protein (1) and fusion protein (2) respectively: (1) NLS-TALE-GCN4 or ERT2-TALE-GCN4 or (ERT2)n-TALE-GCN4, (2)ScFv-ERT2-ADs or ScFv-(ERT2)n-ADs. Based on ERT2 fusion, the 4-OHT-induced TALE transcriptional activation system is achieved.This system is helpful to control the dynamic biological process effectively and precisely, and has great value for the research and development of biomedicine.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a drug-induced gene transcription activation method. Background technique [0002] From targeted genome editing to transcriptional regulation, TALE proteins have been widely used in different fields. Tal protein includes a series of repeat regions, each repeat region contains 33-34 amino acids. The repeat variable diresidues (RVDs) in the middle determine the first base to bind. The simple one-to-one "Tal code" is linked together to determine the specificity of Tal array binding to DNA, so when TAL is fused with effectors related to gene or epigenetic regulation, it can target specific gene loci to play a role . For example, when TALE is fused with a transcriptional activation domain (activator domain, AD) such as VP64, the Tal array targeting the promoter sequence can mediate specific gene transcriptional activation. [0003] Many biological processes are regulated by highly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/62C07K19/00
CPCC07K14/4702C07K2319/00C12N15/85
Inventor 王宇赵晨张月
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI