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Master's Dissertation
DOI
10.11606/D.87.2010.tde-18062010-125910
Document
Author
Full name
Anna Carolina Pereira Vieira de Carvalho
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Strauss, Bryan Eric (President)
Jasiulionis, Miriam Galvonas
Sacramento, Chester Bittencourt
Title in Portuguese
Construção e caracterização de um vírus Adeno-associado com expressão direcionada para células em divisão
Keywords in Portuguese
Divisão celular
Expressão gênica
Neoplasias
Terapia biológica
Transferência de genes
Vetores
Abstract in Portuguese
A utilização do vírus adeno-associado recombinante (AAVr) como vetor de transferência gênica em células tumorais está crescendo. Neste trabalho, o promotor gênico de E2F-1, um promotor ativo durante a divisão celular, foi inserido no AAVr e utilizado para dirigir a expressão do HSV-tk ou luciferase e, simultaneamente, eGFP afim de direcionar a expressão viral para células em proliferação. Em paralelo, foram construídos vetores portadores do promotor constitutivo CMV para servir como controles. O promotor gênico de E2F-1 não foi eficiente em dirigir a expressão dos transgenes na linhagem celular HT1080, enquanto o promotor CMV apresentou uma alta expressão dos repórteres e do gene terapêutico. A baixa eficiência do promotor E2F-1 ainda não foi explorada, mas poderia ser relacionada com o desempenho intrínseco deste promotor, a biologia do vetor AAVr e especificidade celular. Contudo, o bom desempenho do vetor AAVr contendo o promotor CMV abre a possibilidade de realizar novos ensaios de transferência gênica para tratamento e visualização de células tumorais
Title in English
Construction and characterization of adeno-associated virus with limited expression for proliferating cells
Keywords in English
Biology therapy
Cell division
Gene expression
Gene transfer
Neoplasm
Vector
Abstract in English
The utilization of recombinant adeno-associated virus (AAVr) as a gene transfer vector in tumor cells is increasing. In this work, the promoter of the E2F-1 gene, active during cell division, was inserted in an AAVr vector and used to drive the expression of HSV-tk or luciferase and, simultaneously, eGFP with the intent of limiting viral expression to proliferating cells. Also, vectors with the constitutive CMV promoter were constructed to be used as controls. The E2F-1 promoter was not efficient in driving the expression of the transgenes in the HT1080 cell line, while the CMV promoter shows high level expression of the reporter and the therapeutic genes. The low efficiency of E2F promoter has not yet been explored, though this problem could be related to the intrinsic performance of this promoter, the biology of the vector AAV and cell-specific factors. However, the performance of the AAVr containing the CMV promoter creates the possibility of performing new gene transfer protocols for the treatment and visualization of tumor cells.
 
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Publishing Date
2010-09-16
 
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