Induced slow virus expression system and construction method and application thereof

An expression system and construction method technology, applied in the field of functional genomics research, can solve problems such as limited effect, and achieve the effects of sensitive response, small molecular weight and high efficiency

Active Publication Date: 2016-02-17
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on the above, the researchers are committed to the improvement of rtTA, and the new system developed to a certain extent reduces the leaky expression of the induction system and improves the induction efficiency, but the effect is limited

Method used

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  • Induced slow virus expression system and construction method and application thereof
  • Induced slow virus expression system and construction method and application thereof
  • Induced slow virus expression system and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Vector construction and functional analysis of different inducible promoters and antisense tetracycline transcriptional activators

[0046] In this example, two inducible promoters, TetO6 and TRE3G, were combined with two antisense tetracycline transcriptional activators, rtTA3 and TetON3G, for systematic comparison and analysis, and the four combinations (TetO6-rtTA3, TetO6-TetON3G, TRE3G-rtTA3, TRE3G-TetON3G) participates in the induced expression level and background expression level of the target gene of the corresponding inducible vector. Wherein the preparation steps are as follows:

[0047] (1) Transformation of pLVX-Puro vector

[0048] The pLVX-Puro (Clontech) vector was selected as the lentiviral backbone vector. First, the Tth111I, SanII, and BsmBI sites on the Puro sequence were mutated by site-directed mutagenesis, and then the pLVX-Puro vector was used as a template, and the upstream primer (SEQ ID NO: 7 ) and downstream primers (SEQ ID NO: 8) ...

Embodiment 2

[0070] Example 2 Construction and functional analysis of inducible vectors with different insertion directions

[0071] In this example, the positive and negative insertion directions of the target gene expression cassette were compared and analyzed, and the effects of the positive and negative insertion directions on the induction system were analyzed by inducing expression efficiency and background leakage level.

[0072] (1) Construction of reverse induction expression vector

[0073] Using the pLVX-TetO6-RFP-EF1a-TetON3G carrier as a template, use the upstream primer (SEQIDNo: 15) and the downstream primer (SEQIDNo: 16) to amplify the TetO6-RFP fragment; use the pLVX-TRE3G-RFP-EF1a-TetON3G carrier as The template was amplified with an upstream primer (SEQ ID No: 17) and a downstream primer (SEQ ID No: 16) to obtain a TRE3G-RFP fragment. Digest the PCR product with EcoRI-XbaI, and then clone it into the pLVX-EF1a-TetON3G or pLVX-EF1a-rtTA3 vector that has been digested by ...

Embodiment 3

[0084] Construction and functional analysis of inducible vectors of different promoters of embodiment 3

[0085] This example systematically studies the effects of five different antisense tetracycline transcription activator promoters on the system induction efficiency, and further analyzes the gene induction of the vector by the expression level of tetracycline transcription activator on the basis of the screened vector skeleton structure Effect of expression level and background dropout level.

[0086] (1) Construction of different promoter vectors

[0087] With pIRESneo-FLAG-HA-Ago2 (Addgene) vector as template, use upstream primer (SEQIDNo: 18) and downstream primer (SEQIDNo: 19) to amplify the CMV fragment; with pLVX-AmCyan1-C1 (Clontech) vector as template, use Upstream primers (SEQIDNo: 20) and downstream primers (SEQIDNo: 21) amplify the PGK fragment; using the pTRIPz (Thermofisher) vector as a template, use the upstream primer (SEQIDNo: 22) and the downstream primer...

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Abstract

The invention discloses an induced slow virus expression system which comprises a target gene expression cassette and an rtTA expression cassette. The target gene expression cassette comprises an induced promoter containing a tetracycline cis-acting response element and a target gene, and the rtTA expression cassette comprises a promoter, an rtTA, a linker peptide and a screening gene, wherein a P2A linker peptide is adopted as the linker peptide, the screening gene is Puro, and the induced promoter is TetO6 or TRE3G. The invention further discloses a construction method of the induced slow virus expression system. The construction method comprises the steps of pLVX-Puro carrier reconstructing; TetON3G gene synthesizing and cloning; pLVX-rtTA3 constructing; EF1a promoter subcloning; red fluorescence protein (RFP) subcloning; subcloning of the induced promoter TRE3G or TETO6 containing the tetracycline cis-acting response element. The induced slow virus expression system can carry out inducible expression of the target gene to the greatest level while keeping low background expression after cells are introduced into the system, is sensitive in response for an inducer, high in efficiency and low in molecular weight and can be widely applied to gene function research.

Description

technical field [0001] The invention belongs to the field of functional genomics research, and in particular relates to an inducible lentiviral expression system and its construction method and application. Background technique [0002] Overexpression technology is a common method to study gene function, and inducible overexpression is to precisely control the induced expression of the target gene by adding inducers. It is a powerful tool for studying gene function, especially in transgenic animals. . [0003] There are four types of inducible expression systems that have been developed so far: Cre-loxP system (cre-loxp recombinase system), tetracycline (Tet) inducible system, ecdysone inducible system and lactose inducible system. Among them, the tetracycline-induced regulation system is the most widely used induction expression system, which is divided into two categories: tTA system (Tet-off) and rtTA system (Tet-on). Among them, Tet-on has been widely used in transgeni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66
Inventor 苟德明康康黄炼李洁璇牛燕琴张利敏吴伊可姚丽君
Owner SHENZHEN UNIV
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