Pseudo-attP site based integrated general-purpose expression vector and construction method and application thereof

An expression vector and a general-purpose technology, applied in the field of genetic engineering, can solve the problems of time-consuming and heavy workload, and achieve the effect of avoiding gene expression silencing and other potential safety hazards.

Active Publication Date: 2013-08-28
NORTHWEST A & F UNIV +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, this series of conventional detection methods is a huge workload and takes a lot of time

Method used

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  • Pseudo-attP site based integrated general-purpose expression vector and construction method and application thereof
  • Pseudo-attP site based integrated general-purpose expression vector and construction method and application thereof
  • Pseudo-attP site based integrated general-purpose expression vector and construction method and application thereof

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Embodiment Construction

[0045] The universal expression vector based on the integration of the false attP site provided by the present invention can be site-specifically integrated into the false attP site in the genome of eukaryotic cells under the mediation of Streptomyces phage φC31 integrase, thereby Make the transgene express continuously and efficiently, and solve the problems of vector integration site and transgene expression level; then, after positive cells are obtained through drug screening, the antibiotic selection marker is excised under the action of Cre recombination and cleavage, and the excision efficiency is detected by a dual fluorescent reporter system Finally, the transgenic cells that are completely excised from the screening markers are sorted by flow cytometry, and used for somatic cell nuclear transfer to produce transgenic animals without antibiotic screening markers, thereby improving the safety of transgenic animals and providing valuable information for animal transgenic r...

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Abstract

The invention discloses a pseudo-attP site based integrated general-purpose expression vector and a construction method and application thereof. The expression vector comprises an attB sequence, pUC ori and multiple cloning sites MCS, wherein a CMV (cytomegalovirus) constitutive promoter is connected with the attB sequence, and two equidirectional LoxP elements are inserted into the backwards position of the CMV constitutive promoter; and a first fluorescent indicator protein open reading frame and a terminator thereof are arranged at the backwards positions of the LoxP elements, and a secondfluorescent indicator protein open reading frame and a terminator thereof and antibiotic expression elements are arranged between the two LoxP elements. The vector can be used to screen recombinant cells stably expressing fluorescently-marked proteins without antibiotic screening markers.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a site-specifically integrated expression vector, in particular to a universal expression vector based on pseudo attP site integration and its construction method and application. Background technique [0002] Research on transgenic animals is now a hot spot in scientific research. Through transgenic technology, people can break the interspecies isolation to obtain economic animals with excellent traits, can obtain various biological materials and medicinal proteins through the production of bioreactors, can break immune rejection for organ transplantation, and can establish animals for various human diseases Models and analyzes to study specific gene function. However, the existing transgenic animal production technology still has certain drawbacks: the position effect caused by the random insertion of foreign genes, that is, the random insertion of foreign genes into genes rela...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63
Inventor 张涌余源郭泽坤王勇胜田进海胡广东苏峰刘军
Owner NORTHWEST A & F UNIV
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