Generation of cd63-deficient zebrafish to analyze the role of cd63 in viral infection

다국어 초록 (Multilingual Abstract)

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys- Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

목차 (Table of Contents)

• ABSTRACT
• 1. Introduction
• 2. Materials and methods
• 2.1. Zebrafish maintenance
• 2.2. Identification and in silico analysis of Cd63
• 2.3. Whole mount in situ hybridization
• 2.4. Infection of zebrafish with VHSV
• 2.5. Sample collection
• 2.6. RNA isolation and synthesis of complementary DNA
• 2.7. Calculation of VHSV copy number
• 2.8. Generation of cd63 knockout zebrafish using CRISPR/Cas9 technology
• 2.9. Gene expression analysis using qPCR
• 3. Results and discussion
• 3.1. In silico analysis of zebrafish Cd63
• 3.2. Expression patterns of cd63 in zebrafish
• 3.3. Relative mRNA expression of cd63 in zebrafish upon VHSV stimulation
• 3.4. Generation of cd63 knockout mutants using CRISPR/Cas9 gene editing
• 3.5. Analysis of cd63-related gene expression in mutants
• 4. Conclusion
• References