Optimization of the in vitro production system of pig embryos as a basis for the generation of a genetically modified pig model

• Grant number: 22/06031-4
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: August 01, 2022
• End date: April 30, 2023
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Andréa Cristina Basso
• Grantee: Andréa Cristina Basso
• Company: Salt Biotechnology Ltda
• CNAE: Pesquisa e desenvolvimento experimental em ciências físicas e naturais ; Atividades veterinárias
• City: Barretos
• Associated scholarship(s): 22/10948-0 - Optimization of the in vitro production system of pig embryos as a basis for the generation of a genetically modified pig model, BP.PIPE

Abstract

In vitro embryo production (IVP) is a reproduction biotechnique used commercially and on a large scale only in bovine species, despite the possibility of application in several species. In the swine species, IVP has been used basically in research, in order to improve basic knowledge in reproductive physiology. However, the effective application in this species allows several opportunities, such as the production of genetically edited swine embryos for the study of human diseases. In order to be successful in the in vitro production of embryos and, later, in the generation of genetically edited animals, it is essential to master all stages of IVP, namely: 1) in vitro maturation of oocytes (IVM); 2) in vitro fertilization (IVF); 3) in vitro embryo culture (IVC); and 4) embryo cryopreservation. Pig IVP has advanced significantly in the last decade, however, the incidence of chromosomal abnormalities resulting from the high rates of polyspermy together with the inefficiency of the available culture media are pointed out as the main impasses to standardize and increase the rates of embryonic development, as well as as the quality of the embryos generated in the laboratory. Currently, swine IVP lacks specific, validated and commercially available culture media for this purpose. Thus, we aim to develop and validate culture media for the entire in vitro production process of swine embryos, from oocyte maturation to embryo freezing. Furthermore, through the development and validation of more efficient and standardized protocols, together with ideal culture media for the production of swine embryos, the SALT BIOTECH company, in addition to adding a new product to its portfolio, will also be able to fully capitalize on the power of new methods, such as the generation of genetically modified pigs. For this, thinking of a first model, we also propose to develop in silico the components necessary for the future production of an innovative swine model for the study of bladder cancer, in which the known tumor suppressors PTEN and CDKN2A will be knocked out. The project will be carried out in its entirety at SALT BIOTECH and the proposed work will be divided into 2 experiments: production and validation of culture media for swine IVP and in silico design and validation of CRISPR plasmids and guide RNAs to perform the knockout of target genes. (AU)