Production and characterization of multipotent and induced pluripotent cells in Blastocerus dichotomus

• Grant number: 14/02439-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: September 09, 2014
• End date: September 07, 2015
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: José Maurício Barbanti Duarte
• Grantee: Luciana Diniz Rola
• Supervisor: Lawrence C. Smith
• Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil
• Institution abroad: Université de Montréal, Canada
• Associated to the scholarship: 13/13972-0 - Production and characterization of multipotente and induced pluripotent cells in Blastocerus dichotomus, BP.DR

Abstract

Cryopreservation of gametes and embryos is extremely important for maintenance of the genetic variability in small wild populations; however these materials are of difficult collection. Studies of induced pluripotent stem cells (iPS) in human and mouse have showed that cells are capable to differentiate into germ cells, sperm, and oocytes. In cervids, studies regarding iPS are inedit, thus our focus on this project is to establish protocols for obtaining stable iPS lines in Blastocerus dichotomus species to produce viable gametes in the future. This project propose the derivation of iPS from multipotent lines (fat and antler) and comparison with the unipotent line (skin), which will be conducted by means of gene transduction using the PiggyBac tool. The cultures for derivation of iPS will be carried out in the presence of mouse embryonic fibroblast (MEF) blocked by mitomycin, and the selected colonies will be subcultured to maintain lines for further characterization by immunocytochemistry, RT-qPCR, alkaline phosphatase activity, lacZ dye, karyotype, ability to form embryoid bodies, cells differentiate into germline of females, evaluation of markers of tissues derived from the three germ layers, and in vitro differentiation in immunodeficient mice to assess the formation of the three germ layers. Thereafter, tests will be performed aiming to derive iPS into female and male gametes. (AU)