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Cloning of cone-specific inducible expression using CRISPR Cre-Lox system

Grant number: 23/11449-0
Support Opportunities:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): January 01, 2024
Effective date (End): June 30, 2024
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Alexandre Hiroaki Kihara
Grantee:Théo Henrique de Lima Vasconcellos
Supervisor: David E. Cobrinik
Host Institution: Centro de Matemática, Computação e Cognição (CMCC). Universidade Federal do ABC (UFABC). Ministério da Educação (Brasil). Santo André , SP, Brazil
Research place: University of Southern California (USC), United States  
Associated to the scholarship:21/11969-9 - The role of IP3R1 in neuroinflammation triggered by retinitis pigmentosa, BP.MS


Retinal organoids (ROs) provide a remarkable platform for studying retinal development and diseases. These self-organized structures resemble native retinal tissue, encompassing various cell types and functioning photoreceptors. ROs enable the modeling of retinal diseases using patient-derived cells, induced pluripotent stem cell (iPSC), offering insights into disease mechanisms and potential therapeutic strategies. This project aims to delve into advanced gene editing techniques combining CRISPR/Cas9 and Cre-Lox systems. CRISPR/Cas9 offers precise DNA modification, while the Cre-Lox system facilitates controlled gene activation. The focus is developing an inducible Cre-Lox system in cone photoreceptors, enabling specific gene activation using tamoxifen. The primary objective of this project is to integrate the estrogen receptor T2 (ERT2)-Cre-ERT2 cassette into the guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2) gene location and the CAG-LSL-GOI-T2A-Maroon sequence into the AAVS1 gene location of iPSCs. Subsequently, the focus will be on differentiating these iPSC lines into retinal organoids. Previous results from the laboratory abroad where this project will be developed revealed that in retinal organoids generated from iPSCs the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cone photoreceptors. This approach holds potential for disease modeling that affects cones. In summary, the project provides a deep understanding of retinal morphology, cellular interactions, and gene editing techniques. It highlights the potential of retinal organoids and inducible genetic systems for advancing our understanding of retinal development, disease mechanisms, and potential treatments. (AU)

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