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

Grant number: 13/13972-0
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): October 01, 2013
Effective date (End): February 28, 2017
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:José Maurício Barbanti Duarte
Grantee:Luciana Diniz Rola
Home Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil
Associated scholarship(s):14/02439-2 - Production and characterization of multipotent and induced pluripotent cells in Blastocerus dichotomus, BE.EP.DR

Abstract

Cryopreservation of gametes and embryos is extremely important for maintenance of genetic variability in small wild populations, however these materials are difficult to obtain. Studies with human and mouse induced pluripotent stem cells (iPS) have showed that cells are capable to differentiate into germ cells, sperm and oocytes. Studies with iPS in cervids are novel, so we have as focus on this project to establish protocols for obtaining stable iPS lines in Blastocerus dichotomus species to produce viable gametes in the future. Reprogramming of somatic cells into iPS cells is facilitated by the use of adult stem cells and, because little is known about the multipotent stem cells in cervids, the first phase of the project aims to obtain biopsies from various tissues (adipose tissue, dental pulp, antler button), that will be compared with unipotentes cell lines derived from skin biopsies (negative control). The viability of the cells will be assessed with trypan blue, and markers of mesenchymal cells will be assessed by RT-qPCR. Subsequently, these tissues will be cryopreserved and your viability after thawing will be tested. The second phase will focus in the derivation of iPS cells from multipotent lines of the preceding phase, which is conducted by means of gene transduction using the PiggyBac tool. The cultures for derivation of iPS will be made in the presence of mouse embryonic fibroblast (MEF) blocked by mitomycin, and colonies selected 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.

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
BUZANSKAS, MARCOS ELI; GROSSI, DANIELA DO AMARAL; VENTURA, RICARDO VIEIRA; SCHENKEL, FLAVIO SCHRAMM; SELEGUIM CHUD, TATIANE CRISTINA; STAFUZZA, NEDENIA BONVINO; ROLA, LUCIANA DINIZ; CONCEICAO MEIRELLES, SARAH LAGUNA; MOKRY, FABIANA BARICHELLO; MUDADU, MAURICIO DE ALVARENGA; HIGA, ROBERTO HIROSHI; GUALBERTO BARBOSA DA SILVA, MARCOS VINICIUS; DE ALENCAR, MAURICIO MELLO; DE ALMEIDA REGITANO, LUCIANA CORREIA; MUNARI, DANISIO PRADO. Candidate genes for male and female reproductive traits in Canchim beef cattle. JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY, v. 8, AUG 23 2017. Web of Science Citations: 6.
BUZANSKAS, M. E.; PIRES, P. S.; CHUD, T. C. S.; BERNARDES, P. A.; ROLA, L. D.; SAVEGNAGO, R. P.; LOBO, R. B.; MUNARI, D. P. Parameter estimates for reproductive and carcass traits in Nelore beef cattle. Theriogenology, v. 92, p. 204-209, APR 1 2017. Web of Science Citations: 10.
Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
ROLA, Luciana Diniz. Produção e caracterização de células multipotentes e pluripotentes induzidas em Blastocerus dichotomus. 2017. Doctoral Thesis - Universidade Estadual Paulista "Júlio de Mesquita Filho" Faculdade de Ciências Agrárias e Veterinárias..

Please report errors in scientific publications list by writing to: cdi@fapesp.br.