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New hepatic cell lines for research, drug testing and vaccine development: Molecular tools for high-throughput screening of HCV replicating cells
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Orientador(es)
Resumo(s)
Hepatitis C virus (HCV) is a main contributor to chronic liver disease, causing a major social and economic burden worldwide. There is a demand for the development of a vaccine to prevent transmission and eliminate HCV infection. This development has been limited by the lack of research and improved tools, including in vitro hepatic cell model permissive to HCV wild-type infection.
This work aimed at developing and validating the molecular tools for a high-throughput screening of highly competent hepatic cell lines in supporting the HCV life-cycle, and implementing and optimizing protocols to produce and handle HCV. This molecular tool consisted of a full-length replicon based on the J6/C tagged with a GFP reporter, enabling to identify HCV permissive cells. Since the tagged replicon consists of a RNA molecule, we started by optimizing method for delivering into Huh-7.5 cells, using Lipofectamine MessengerMAX, and by implementing an in vitro transcription (IVT) protocol. Additionally, we establish reporter plasmids to produce control transcripts, suiting the purpose of IVT validation and to function as a transfection internal control. As a mechanism for translation initiation in these reporter constructions, two possibilities were evaluated: a cap-dependent mechanism, using ARCA, and a capindependent mechanism, based on four types of IRES. After establishing the protocols to produce and deliver the tagged replicon into cells, the functionality of this RNA construction was confirmed by the reporter gene expression, when transfected in Huh-7.5 cell line. After the validation of the reporter capacity of the tagged replicon, to continuo in the follow-up of this thesis, new hepatic cell lines established by immortalization from primary human hepatocytes will be assessed their ability to support the HCV life cycle.
This work contributed to create a methodology for screening improved hepatic cell lines to better serve research, drug testing and vaccine development against HCV.
Descrição
Palavras-chave
HCV Tagged replicon Lipofection In vitro transcription human hepatic cell lines