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Eukaryotic cell system for detecting integrase phi-C31 functions and preparation method thereof

A technology of eukaryotic cells and integrase, applied in the field of biotechnology and gene therapy, can solve the problems of large errors and complicated operation steps, and achieve the effect of high accuracy, good repeatability and few steps

Inactive Publication Date: 2006-09-06
FUDAN UNIV
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Problems solved by technology

This method can quantitatively explain the function of integrase ΦC31 in mouse cells, but the operation steps are complicated and the error is relatively large

Method used

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  • Eukaryotic cell system for detecting integrase phi-C31 functions and preparation method thereof
  • Eukaryotic cell system for detecting integrase phi-C31 functions and preparation method thereof
  • Eukaryotic cell system for detecting integrase phi-C31 functions and preparation method thereof

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Embodiment Construction

[0015] The present invention will be further described below in conjunction with specific implementation examples. It should be understood that these implementation examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions such as Sambrook et al. (Guide to Cell Experiments, Laboratory Manual, Second Edition, 1998, Science Press), or in accordance with the conditions suggested by the manufacturer.

[0016] 1. Construct the recombinant reporter plasmid PB[EGFP]:

[0017] Through conventional DNA recombination techniques, the construction of the recombinant reporter plasmid PB[EGFP] follows the following steps:

[0018] First clone attB into the pEGFP-N2 vector (product of Clontech Company), using EcoRI-5'-ACCGGAATTCGTCGACATGCCCGCC-3' and KpnI-5'-AATAACGGTACCGTCGACGATGTAGGT...

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Abstract

The invention relates the eukaryotic cell system and method for checking integrated enzymeªÁC31 function, belonging to biotechnology and causal treatment technology field. The method comprises the following steps: after recombination report PB [EGFP] transfer dyeing the embryon kidney cell system, screening checking system, choosing monoclonal cell, getting the product, the PB[EGFP] using the pEGFP-N2 as carrier, after enhancing CMVIE, sequence inserting specificity sequence attP and attB, nd then adding growth factor polyadenylic acid. The cell system can estimate the integrated enzymeªÁC31 expressed by transfer dyeing, transduction and conversion approach, and the activity and function of its mutant. The checking system has the advantages of simple operation, high accuracy, and good repeatability. The system is the important quantitative estimation system that integrated enzymeªÁC31 is in the application of mammalian cytology.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and gene therapy, and specifically relates to a eukaryotic cell system for detecting the function and activity of integrase ΦC31 in mammalian cells and a preparation method thereof. Background technique [0002] Phage integrase ΦC31 can site-directly integrate foreign nucleotide sequences into specific sites in mammalian cell genomes, and has become a useful tool for transgenic site-specific recombination operations and gene therapy in mammalian cells in recent years. [0003] In 2000, Calos et al. first carried out the study of site-directed integration in mammalian cells using phage integrase ΦC31, and then realized the gene therapy of coagulation factor IX mediated by integrase ΦC31 in the animal model of mice. The construction of high-efficiency transgenic animals has been realized in flies and other animals, thus opening up a new direction of efficient site-specific genome integration r...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张茂祥朱焕章陈金中王韧诚贾韦国薛京伦
Owner FUDAN UNIV
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