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Doctoral Thesis
DOI
10.11606/T.87.2016.tde-24082016-113658
Document
Author
Full name
Juliana Fontes Beltran Paschoal
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Jorge, Soraia Attie Calil (President)
Azzoni, Adriano Rodrigues
Durigon, Edison Luiz
Santos, Milene Cristina Menezes dos
Soares, Carlos Roberto Jorge
Title in Portuguese
Expressão gênica empregando pseudopartículas em células de mamíferos (HEK 293T e Huh 7.0) cultivadas em diferentes meios de cultura livres de soro.
Keywords in Portuguese
Adaptação em meios livres de Soro (SFM)
Células de mamíferos
Glicoproteína do vírus rábico (RVGP)
Pseudopartículas virais
Abstract in Portuguese
Células HEK293T e Huh7 foram adaptadas em meios livres de soro fetal bovino (SFM). Parâmetros metabólicos e de crescimento foram avaliados, além da expressão gênica heteróloga, utilizando um sistema de expressão que produz pseudo-partículas (ppHCV), derivadas do vírus da Leucemia Murina (MLV) e da Hepatite C (HCV). A adaptação foi realizada através de diluição sequencial para SFM. A linhagem HEK293T foi adaptada em dois SFM: Hybridoma-SFM e CHO-S-SFMII, a linhagem Huh7 foi adaptada nos quatro SFM escolhidos. O consumo de substratos para cada linhagem foi diferente entre os SFM, apesar de o crescimento celular ter sido semelhante. Para a análise da expressão gênica, três vetores foram co-transfectados em células HEK293T. Foi observado que para a produção de ppHCV, o tempo de coleta foi de 48 horas. O método de co-transfecção por lipofectamina produziu mais cópias de vírus, sendo que quantificações de 5,30x103 cópias RNA/μL foram encontradas para vírus produzidos em células adaptadas no meio Hybridoma-SFM através de qRT-PCR. Estas ppHCV foram usadas para infectar células Huh7, células infectadas produziram cerca de 10 ng de proteína recombinante/106 células.
Title in English
Gene expression using pseudoparticles in cultured mammalian cells (HEK 293T and Huh 7.0) in diferent serum free medium.
Keywords in English
Adaptation in serum free media (SFM)
Glycoprotein of the rabies virus (RVGP)
Mammalian cell culture
Viral pseudoparticles
Abstract in English
HEK 293-T and Huh7 cells were adapted in serum free mediu (SFM). Metabolic and growth parameters were assessed, as well as heterologous gene expression, using an expression system that produces pseudo-particles (ppHCV), derived from the murine leukemia virus (MLV), and Hepatitis C (HCV). The adaptation was performed by sequential dilution in SFM. The HEK- 293T line was adapted in two SFM: Hybridoma-SFM and CHO-S-SFMII, the Huh7 line was adapted in four chosen SFM. The consumption of substrates were different for each line in SFM, while cell growth was similar. For the analysis of gene expression, three vectors were co-transfected into HEK-293T cells. It was observed that for the production of ppHCV, the collection time was 48 hours. The method of co-transfection with lipofectamine produced more copies of the virus into the cells, 5,30 x103 RNA copies/μL were found to virus produced in the cells adapted in Hybridoma- SFM, by qRT-PCR. These ppHCV were used to infect Huh 7, infected cells produced around 10 ng recombinant protein /106 cells.
 
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Publishing Date
2016-08-24
 
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