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Insertion of fluorescent reporter gene in Sox2 gene region to study cell differentiation in early bovine embryo development

Grant number: 19/03014-9
Support type:Scholarships in Brazil - Master
Effective date (Start): June 01, 2019
Effective date (End): October 31, 2021
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal researcher:Marcelo Demarchi Goissis
Grantee:Felipe Eduardo Luedke
Home Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:17/09576-3 - Studies on cell differentiation mechanisms during early bovine embryo development, AP.JP

Abstract

The segregation between the inner cell mass (MCI) and the trophectoderm (TE) is the first event of cellular differentiation that occurs in the mammalian embryo. A coordinated expression of many genes occurs in these early events. Failures in these early developmental processes can cause embryonic and consequently, economic losses. Studies on cell differentiation in cattle are still scarce, reflecting in low success rates in the production of in vitro bovine embryos. Techniques for studying embryos usually involve their destruction and impair real-time monitoring of the dynamics of gene expression related to MCI and TE differentiation. New techniques such as CRISPR/Cas9 allow directed alterations of the genomic DNA sequence. The targeted introduction of fluorescent protein reporters would allow real-time visualization of gene expression in bovine embryos. It is intended to perform gene editing in the region of the Sox2 gene for the introduction of a fluorescent protein by homologous recombination. It is also intended to compare the efficiency of gene editing at different stages of embryonic development. The experiments will involve techniques of molecular cloning, microinjection of embryos and fluorescence microscopy. The aim of this project is to develop a tool for targeted introduction of reporter genes into bovine embryos and at the same time, to understand the mechanisms of cell differentiation, contributing to the improvement of in vitro embryo production protocols. (AU)

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