RESUMO
Spermatogonial stem cells (SSCs), the unique seed cells of testes, can undergo meiosis and form spermatozoa, thus transmitting genetic information to offspring. Research concerning these cells explores the mechanism underlying spermatogenesis, making possible the induction of their differentiation into spermatozoa in vitro. SSCs have therefore attracted much interest among scientists. Although the proliferation of such cells in vitro has been demonstrated, we are unaware of any long-term laboratory culture of porcine SSCs. The objective of this study was to isolate, characterize, culture, and induce the differentiation of Bama mini-pig SSCs. SSCs were isolated using differential plating and cultured for over 100 days on an STO feeder cell layer without serum. Cell clusters appeared after three passages and continuously formed during subsequent cultivation. Staining showed that these clusters were positive for UCHL1 and CDH1, could be bound by Dolichos biflorus agglutinin, and that some cells expressed OCT4. Ultrastructure observations revealed SSCs in testis tissue to be round in shape, while those cultured in vitro were flat and bound together. Our attempts at inducing differentiation showed that SSCs cultured in vitro could undergo meiosis. In this study, we describe an effective culture system for Bama mini-pig SSCs capable of producing enough cells to establish a platform for further SSC research, such as genetic manipulation or exploration of the mechanism underlying spermatogenesis.
Assuntos
Espermatogônias/citologia , Células-Tronco/fisiologia , Animais , Biomarcadores/metabolismo , Agregação Celular , Diferenciação Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Masculino , Meiose , Espermatogênese , Suínos , Porco Miniatura , Testículo/citologiaRESUMO
Considerable attention is currently being directed toward methods for producing recombinant human proteins in the mammary glands of genetically modified transgenic livestock. However, the expression of inserted genes in transgenic animals is variable and often very low because of the randomness of the site of transgene integration. One possible strategy to avoid the expression problem associated with random integration is to use site-specific integration by targeting integration to a high expression locus and, thereby, to improve expression of the transferred gene. In the present study, we focused on glial cell line-derived neurotrophic factor (GDNF), a novel type of neurotrophic factor first cloned in 1993. Research has shown that GDNF may have potential applications in the treatment of Parkinson's disease and other diseases of the central nervous system since it acts as a protective factor for central dopaminergic neurons. Here, we constructed a gene targeting vector to knock-in the human GDNF gene at the bovine beta-casein gene locus as a first step to producing transgenic animals with a high level of expression of human GDNF protein in their mammary glands. Bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation. Three cell clones were identified with successful targeting to the beta-casein locus; and were confirmed using both polymerase chain reaction analysis and sequencing. Gene-targeted cells were used as nuclear donors; a total of 161 embryos were reconstructed, 23 of which developed to the blastocyst stage. These blastocysts were transferred to 8 recipient cows, but no offspring were obtained.
Assuntos
Caseínas/genética , Bovinos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Animais , Animais Geneticamente Modificados , Blastocisto , Bovinos/metabolismo , Feminino , Marcação de Genes , Genes Reporter , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , TransfecçãoRESUMO
The c-kit protein plays a major role in the regulation of germ cell development. Its expression and distribution in rodent testes have been widely reported. However, research regarding c-kit expression in domestic animals is scarce, and the expression pattern and distribution of c-kit in germ cells have not been clearly defined. In this study, a specific antigenic region for goat c-kit was designed, and a c-kit polyclonal antibody was prepared. This antibody was then applied in a study evaluating c-kit expression in Cashmere goat tissues. A Western blot analysis showed that three forms of c-kit were expressed in goat testes: precursor, mature, and soluble c-kit. Fluorescent immunohistochemical analyses showed that c-kit was primarily expressed in the spermatogonia and spermatocytes of goat testes. These results not only clarify the expression and localization of c-kit in the goat testis, but also accelerate further research regarding the function of c-kit in goat spermatogenesis.