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Tetracycline slow virus inducible expression vector as well as building method and application thereof

A technology of inducing expression and tetracycline, applied in the field of functional genomics research, can solve problems such as imperfection and expression leakage

Active Publication Date: 2017-12-29
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the tetracycline induction system has been greatly developed, the Tet-On system relying on the lentiviral vector is not perfect, because the LTR promoter / enhancer derived from the lentiviral vector can cause expression leakage, especially when constructing the same vector When co-expressing inducible genes and rtTA, it is more necessary to optimize the best combination

Method used

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  • Tetracycline slow virus inducible expression vector as well as building method and application thereof
  • Tetracycline slow virus inducible expression vector as well as building method and application thereof
  • Tetracycline slow virus inducible expression vector as well as building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Vector Construction and Functional Analysis of Different Inducible Promoters, Induction Directions and Antisense Tetracycline Transcription Activators

[0082] In this example, three inducible promoters, TRE3Gp, TRE3Gs, and TetO6, and two antisense tetracycline transcriptional activators, rtTA3 and TetON3G, were systematically compared and analyzed in different induction directions, and the corresponding inducible vectors formed by 12 combinations were analyzed. The induced expression level, background expression level and corresponding induction fold of the target gene.

[0083] (1) Transformation of pLVX-Puro vector

[0084] This system uses pLVX-Puro (Clontech) vector as the lentiviral backbone vector. First, the Tth111I, SacII, and BsmBI sites on the Puro sequence were mutated by site-directed mutagenesis, and then the pLVX-Puro vector was used as a template to amplify with the upstream primer (SEQ ID NO: 8) and downstream primer (SEQ ID NO: 9). The Puro...

Embodiment 2

[0118] Example 2 Construction and Functional Analysis of Vectors of Different Antisense Tetracycline Transcription Activator Promoters

[0119] This example quantitatively studies the effects of five different antisense tetracycline transcriptional activator promoters on system induction activity, background expression level and induction fold, and further optimizes the vector on the basis of the vector backbone that has been screened .

[0120] (1) Construction of different promoter vectors

[0121] The primers required for amplifying different vector sequences in this example are shown in Table 3. Using the pIRESneo-FLAG-HA-Ago2 (Addgene) vector as a template, use the upstream primer (SEQ ID No: 23) and the downstream primer (SEQ ID No: 24) to amplify the CMV fragment; use pCDH-CMV-MCS-EF1-copGFP (SBI) vector is template, with upstream primer (SEQ ID No: 25) and downstream primer (SEQ ID No: 26) amplification EF1a fragment; With pTRIPz (Thermofisher) vector as template, wi...

Embodiment 3

[0133] Example 3 Application of Novel Tetracycline Inducible System in CRISPR / Cas9 System

[0134] In this example, the newly developed tetracycline induction system was applied to the CRISPR / Cas9 system to construct a new inducible CRISPR / Cas9 system, and the induction efficiency of the inducible CRISPR / Cas9 system was verified at the cellular level.

[0135] (1) Construction of pL / Cas9-TRE3Gp / PGK-TetON3G vector

[0136] Using the LentiCRISPR v2 (Addgene) vector as a template, the Cas9-Flag sequence was amplified by PCR with the upstream primer (SEQ ID No: 31) and the downstream primer (SEQ ID No: 32), and the PCR product was double-digested with AgeI-XbaI, and then reversed. Cloned into the pL / RFP-TRE3Gp / PGK-TetON3G vector that had been cut with the same double restriction enzymes, a new type of inducible CRISPR / Cas9 vector was obtained and named pL / Cas9-TRE3Gp / PGK-TetON3G.

[0137] (2) Construction of pL / U6-ccdb / Cas9-TRE3Gp / PGK-TetON3G vector

[0138]Using the LentiCRISPR...

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Abstract

The invention discloses a tetracycline slow virus inducible expression vector as well as a building method and application thereof. The tetracycline slow virus inducible expression vector comprises a target gene expression frame and an antisense tetracycline transcription activating factor expression frame; an inducible CRISPR / Cas9 system places a Cas9 protein downstream of an inducible promoter, and places an sgRNA sequence of U6 prompting expression upstream of the whole tetracycline inducing system. The combination effect of TRE3Gp and TetON3G is best; after cells are introduced into the induction system, the expression of a target gene can be induced at the highest level while keeping low background expression. The induction system is sensitive to an induction agent, has high efficiency and a small molecular weight, and can be widely applied to gene function researches.

Description

technical field [0001] The invention relates to the field of functional genomics research, and mainly relates to a tetracycline lentivirus-induced expression vector and its establishment method and application. Background technique [0002] Regulation of gene expression can make the study of gene function more precise. In 1992, Gossen et al. successfully constructed a tetracycline (tetracycline, Tet) eukaryotic cell gene expression regulation system using prokaryotic gene regulatory elements, and used Tet or its derivatives to induce the expression of genes of interest. The tetracycline induction system includes Tet-off and Tet-on systems, in which the Tet-on system utilizes the antisense tetracycline transcription activator (rtTA), which is in a closed state without the inducer doxycycline (Dox), and only The expression of the target gene can be induced rapidly only under Dox stimulation, so the Tet-on system is more favored by people (Gossen M, Freundlieb S et al, Science...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66
CPCC12N15/86C12N2740/15043C12N2800/80C12N2810/10C12N2830/002
Inventor 苟德明黄炼康康
Owner SHENZHEN UNIV
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