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Master's Dissertation
DOI
10.11606/D.42.2008.tde-09092008-113238
Document
Author
Full name
Hélcio Yogi Ono
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Moriscot, Anselmo Sigari (President)
Giannocco, Gisele
Peroni, Cibele Nunes
Title in Portuguese
Transferência gênica in vivo por meio de eletroporação em músculo sóleo de ratos wistar.
Keywords in Portuguese
Eletroporação
Músculo esquelético
Plasmídeos
Proteínas de fluorescência verde
Sóleo
Transferência de genes
Abstract in Portuguese
A proposta deste trabalho é a padronização do método de eletroporação direta (utilizando eletrodos) em músculo sóleo de ratos wistar. Deste processo físico, pudemos variar a voltagem, o número de pulsos e duração do pulso. O objetivo principal foi à busca de um parâmetro ideal onde deveria haver a menor lesão tecidual com a maior quantidade de expressão do GFP. Métodos de análise (fluorescência e histologia) foram utilizados para a determinação da eficiência de transferência de genes, onde se considerou a razão entre as fibras musculares sadias emissoras fluorescência e o número total de fibras sadias. Como resultado, atingimos a taxa de eficiência de (50,3 ± 20%) em transferência de genes, utilizando a voltagem de 25 V, trem de 8 pulsos com duração de 20ms e freqüência de 1Hz. A possibilidade de interferir na função deste músculo abre a perspectiva de se estudar o papel de certos genes na plasticidade muscular esquelética, especialmente no crescimento muscular longitudinal.
Title in English
In vivo gene transfer by electroporation in soleus muscle from wistar rats.
Keywords in English
Eletroporation
Gene transfer
Green fluorescence protein
Plasmid
Skeletal muscle
Soleus
Abstract in English
The use of electrical pulses for gene transfer has been successfully used to reach significant levels of gene expression in vitro and in vivo. Therefore electroporation can be considered an important device to get further insight on biological mechanisms and also to treatment. The aim of the present work is to achieve a high level of gene transfer throughout electroporation in the soleus muscle of Wistar rats. The muscle was surgically accessed, injected with Hyaluronidase and subsequently injected with a plasmid expressing GFP (Green Fluorescence Protein). Observation of fluorescence produced in situ and also HE (Hematoxilin-eosin) preparations revealed that the best gene transfer efficiency (50, 3 ± 20%) was achieved with 25 V and 8 pulses (1Hz) lasting 20ms. These results point to a potential use of electroporation as a tool for investigating cellular and molecular aspects of skeletal muscle plasticity, especially those that can be approached only in soleus muscle, such as longitudinal growth.
 
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Publishing Date
2008-09-11
 
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