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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P255 | DOI: 10.1530/repabs.1.P255

Canine spermatogonial stem cells transfected with GFP reporter gene transplanted in recipient teste mice

Naira Caroline Godoy Pieri1, Aline Fernanda de Souza1, Ana Carolina Furlanetto Mançanares1, Juliana Barbosa Casals1, Kelly Cristine Roballo1, Fabiana Bressan1, Lilian Jesus Oliveira1, Carlos Eduardo Ambrosio2 & Daniele Santos Martins1

1University of Sao Paulo, Pirassununga, Brazil; 2School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.

Introduction: Spermatogonial stem cells (SSCs) characterized by ability to self-renew and proliferated, differentiated, and transmitted genetic information. In canine the first attempt (1999) of xenotransplantation into mice did not successfully produce spermatozoa. However, there are evidence that xenogeneic transplant of testis cells can engraft in host testis, and generate donor derived sperm; it suggests the SSCs transplantation may offer a similarity to transgenesis in the canine model. Our aims were to characterize canine SSCs in vitro and development a xenotransplant assay for SSCs transfected with GFP reporter gene into mice.

Materials and methods: Testicular germ cells were isolated and cultured from prepubertal canines and methods for enrichment culture systems were established. The SSCs were transduced with a GFP reporter gene by a lentiviral vector to target the SSCs in recipient testes. Then GFP-transduced SSCs was transplanted into eight testes of C57BL/6 mice treated with busulfan. At 25 and 90 days after xenotransplantation, four recipient animals were euthanized and number of GFP-expressing in testis was analyzed.

Results and discussion: SSCs canine maintained the expression of CD49f and C-kit for 10 days in culture using our enrichment culture system. For the xenotransplant, a total of 105 cells were injected and 20–43% of these cells engraft the testes. Moreover, the clump-forming canine germ cells GFP+ cells colonized membrane basal of seminiferous tubules of mice. This research was conducted in accordance with the Committee of Ethics of the Faculty of Animal Sciences and Food Engineering FZEA-(USP).