Generation and characterization of the tg(lyz: mCherry) transgenic line in zebrafish.

• Grant number: 24/20460-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2025
• End date: December 31, 2025
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Hernandes Faustino de Carvalho
• Grantee: Raffael Rotta Bindi
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Zebrafish are an excellent model for studying diseases of genetic origin because they share high genetic and molecular similarities with humans. In particular, transgenesis and the study of processes involving hematopoietic cells have been widely studied using this fish as a model. Macrophages are cells that can have embryonic and hematopoietic origins and their development is quite conserved with zebrafish due to the ontogeny of blood. Thus, even if some organs change, they are equivalent for their hematopoietic functions, as is the case of the caudal hematopoietic tissue (CHT) in zebrafish and the fetal liver in humans. In parallel, there has been much discussion about the function of tumor-associated macrophages (TAMs), whether they would be in defense of the body or contributing to the tumor environment due to their functions related to angiogenesis and remodeling of the extracellular matrix. Recently, it was elucidated that macrophages present in the prostate after castration are responsible for inducing apoptosis of epithelial cells. In this context, we intend to create a transgenic zebrafish line that marks macrophages through a tg(lyz: RFP) reporter gene. In this way, we believe that we will be able to conduct future research with this line, such as through xenotransplantation of prostate tumor cells in zebrafish, where, if this line is used, it will be possible to observe the behavior of macrophages in vivo. We believe that the promoter region of the Lysozyme type C (lyz) gene is suitable for directing the expression of the RFP (Red Fluorescent Protein) reporter gene, since it presents specific expression in zebrafish macrophages. Approximately 3 KB upstream of the lyz gene has already been used to generate this transgenic line and showed specific expression in macrophages. However, we first intend to perform in silico analyses to verify these data and determine the possible promoter region. Then, we will produce a construct, using the Tal2 transgenesis technique, to inject into zebrafish embryos and generate a transgenic F0 line. After these fish reach reproductive age, we will cross them with wild type (WT) fish to generate the F1 generation. We will also monitor and acquire photos to analyze the morphology, behavior and migration of the RFP+ cells to confirm that they are macrophages and the specificity of the promoter. In this way, we intend to establish a transgenic lineage tg(lyz: RFP) to continue to be perpetuated in the vivarium and to be used in various studies involving macrophages, including monitoring the biology of xenotransplants.