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Compositions and methods related to controlled gene expression using viral vectors

A viral vector and vector technology, applied in the bidirectional promoter, the living body of the expression system, inducible and reversible expression of the target sequence, the animal model field of the sequence, can solve the problem of low efficiency of producing infectious virus vector particles and so on

Inactive Publication Date: 2009-03-11
UAB RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method was found to have many drawbacks, such as the production of infectious viral vector particles with far less than ideal efficiency

Method used

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  • Compositions and methods related to controlled gene expression using viral vectors
  • Compositions and methods related to controlled gene expression using viral vectors
  • Compositions and methods related to controlled gene expression using viral vectors

Examples

Experimental program
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Effect test

Embodiment 1

[0271] Construction of tetracycline-based inducible and reversible single lentiviral vectors

[0272] A tetracycline-based inducible and reversible single gene transfer vector was constructed to drive the expression of the sequence of interest eGFP. First, the 1.2 kb human EF1-a promoter was amplified from pEF4 / His (Invitrogen) by PCR and cloned into pHRCMVeGFP / blas using EcoRI and BamHI restriction enzymes. The resulting vector was named pHREFeGFP / blas. Next, a sequence encoding a tetracycline repressor was codon optimized and linked to the SV40 nuclear localization signal. The gene for the codon-optimized tetracycline repressor linked to the SV40 nuclear localization signal was then cloned into pHREFeGFP / blas using Ncol and XhoI restriction enzymes to replace eGFP. The resulting vector was named pHREFtet / blas. Then, 500 bp of the human CMV promoter was amplified by PCR, and two t et promoter sequences were introduced into the 3' CMV promoter. The PCR fragment was cloned ...

Embodiment 2

[0274] Generation of high titers of tetracycline-based inducible and reversible single virus particles

[0275]293Y cells were co-transfected with packaging constructs, envelope constructs, and different gene transfer constructs (including pHReGFPO2 / EFtet / blas, pHReGFPO2 / CAGtet / blas; pHReGFPO2 / UB6tet / blas, and pHReGFPO2 / CAGtet / blas) to produce different species inducible virus particles. The titers of the viral particles resulting from co-transfection were measured using fluorescence microscopy to determine eGFP expression in HeLa cells. The titer of supernatant from transfected cells was 1-4 x 10 6 / ml, while the titer of the concentrated supernatant was 2-10×10 8 / ml (400 times higher).

Embodiment 3

[0277] Tightly regulated inducible single lentiviral vector

[0278] Use 100 μl derived from pHReGFPO2 / EFtet / blas (titer is 2.5×10 6 / ml) virus particle supernatant to infect mouse T cell line (4×10 4 ). On the next day, the infected cells were divided into the following two groups: the first group, which was cultured in a medium containing 0.1 μg DOX / ml; the second group, which was cultured in a medium without DOX. Three days after infection, the cells were analyzed by FACS analysis to determine the expression level of GFP. Analysis of the first group showed that the average intensity of the GFP expression signal was 16,195, a 44.2-fold increase compared to that of the second group.

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Abstract

The invention provides a method and composition relating to the virus vector. The invention also provides a method and composition for effectively transfecting host (via highly effective slow virus delivery vector), and method for sensitively controlling the expression time and level of transferred target sequence in host by simply giving regulator to host. The invention also discloses a method for preparing transgenic mouse and the transgenic mouse prepared by using virus vector-related composition and method.

Description

[0001] Cross-references to related patent applications [0002] This application claims the benefit of US Provisional Application Serial No. 60 / 751,407, filed December 16, 2005, and US Provisional Application Serial No. 60 / 751,117, filed December 16, 2005. US Provisional Application Serial No. 60 / 751,407, filed December 16, 2005, and US Provisional Application Serial No. 60 / 751,117, filed December 16, 2005 are hereby incorporated by reference in their entireties. [0003] thank you [0004] This invention was made with government support under Grant Nos. R01 AI47717 and R01 AI48852 from the National Institute of Allergy and Infectious Diseases and the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] It is often desirable to transfer and modulate the expression of a sequence of interest in a cell or in a living subject, whether the subject is a cell in culture, or a living subject such as an animal model or a hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12P21/06C12N1/00C12N5/02C12N1/20C07H21/04A61K31/70A01K67/00
CPCC12N15/63C12N15/635C12N2800/30C12N2830/003A01K67/0275A01K2217/052A01K2217/203C12N2740/16043A61P31/18
Inventor X·吴J·卡比斯
Owner UAB RES FOUND
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