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Doctoral Thesis
DOI
10.11606/T.5.2017.tde-14112017-153947
Document
Author
Full name
Rafael Miyashiro Nunes dos Santos
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2017
Supervisor
Committee
Albuquerque, Luiz Augusto Carneiro D (President)
Demasi, Marcos Angelo Almeida
Galvao, Flávio Henrique Ferreira
Silva, Antonio Eduardo Benedito
Title in Portuguese
Substituição gênica ortotópica de porco para humano baseada em CRISPR/Cas9 e recombinases para xenotransplante
Keywords in Portuguese
Linhagem celular
Sistemas CRISPRCas
Técnicas de transferência de genes
Transfecção
Transplante heterólogo
Trombomodulina
Abstract in Portuguese
Modelos humanizados de porco são muito importantes para pesquisa biomédica e desenvolvimento de novas drogas e tratamentos. Além de ser um melhor modelo para doenças humanas do que animais de menor porte devido sua maior semelhança fisiológica, anatômica, de metabolismo e tempo de vida, o modelo suíno ainda permite suprimento ilimitado de órgãos para transplante. Apesar dessas vantagens, a expressão gênica inconsistente de animais transgênicos tornam a criação e avaliação desses animais muito dispendiosas, imprevisível e não permite a comparação de resultados de animais diferentes de maneira apropriada. Nesse estudo descrevemos uma nova técnica utilizando o promoter endógeno para a geração de um protocolo de substituição de genes com padrão clonal (transplante clonal de genes) sem clonagem de células, preservando a expressão genética e sua regulação intactas. Esse protocolo é reprodutível e pode ser aplicado para mais de um alvo genético, permitindo geração rápida de linhas transgênicas de animais (14-20 dias) com potencial de se tornar o novo padrão para geração de animais transgênicos de grande porte Suínos
Title in English
CRISPR/Cas9 and recombinase based pig-to-human orthotopic gene exchange for xenotransplantation
Keywords in English
Cell line
CRISPR-Cas systems
Gene transfer techniques
Thrombomodulin
Transfection swine
Transplantation heterologous
Abstract in English
Humanized pig models are very important for biomedical research, and drugs and treatment development. Not only it is a better model for diseases than smaller animals because of its closer physiology, anatomy, metabolism and life span, it also may provide unlimited organs for transplantation. In spite of all this advantages, inconsistent gene expression in transgenic animals make its generation and evaluation expensive, unpredictable and do not allow proper outcome comparison between different animals. In this report we describe a reproducible technique utilizing the endogenous promoter for generation of a clonal pattern gene replacement protocol (clonal gene transplant) without cell cloning, maintaining the normal gene expression and its regulation. This protocol is reproducible and applicable to more than one gene target, allowing fast generation of transgenic animals cell lines (as low as 14-20 days) and could become the new standard for transgenic large animal generation
 
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Publishing Date
2017-11-16
 
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