RESUMO
Intracytoplasmic sperm injection (ICSI), an important method used to treat male subfertility, is applied in the transgenic technology of sperm-mediated gene transfer. However, no study has described successful generation of offspring using ICSI in the common marmoset, a small non-human primate used as a model for biomedical translational research. In this study, we investigated blastocyst development and the subsequent live offspring stages of marmoset oocytes matured in vitro and fertilized by ICSI. To investigate the optimal timing of performing ICSI, corrected immature oocytes were matured in vitro and ICSI was performed at various time points (1-2 h, 2-4 h, 4-6 h, 6-8 h, and 8-10 h after extrusion of the first polar body (PB)). Matured oocytes were then divided randomly into two groups: one was used for in vitro fertilization (IVF) and the other for ICSI. To investigate in vivo development of embryos followed by ICSI, 6-cell- to 8-cell-stage embryos and blastocysts were nonsurgically transferred into recipient marmosets. Although no significant differences were observed in the fertilization rate of blastocysts among ICSI timing after the first PB extrusion, the blastocyst rate at 1-2 h was lowest among groups at 2-4 h, 4-6 h, 6-8 h, and 8-10 h. Comparing ICSI to IVF, the fertilization rates obtained in ICSI were higher than in IVF (p>0.05). No significant difference was noted in the cleaved blastocyst rate between ICSI and IVF. Following the transfer of 37 ICSI blastocysts, 4 of 20 recipients became pregnant, while with the transfer of 21 6-cell- to 8-cell-stage ICSI embryos, 3 of 8 recipients became pregnant. Four healthy offspring were produced and grew normally. These are the first marmoset offspring produced by ICSI, making it an effective fertilization method for marmosets.
Assuntos
Callithrix/embriologia , Diferenciação Celular , Oócitos/metabolismo , Injeções de Esperma Intracitoplásmicas/métodos , Animais , Animais Recém-Nascidos , Blastocisto/citologia , Transferência Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Marcadores Genéticos , Humanos , Masculino , Repetições de Microssatélites/genética , Corpos Polares/citologia , GravidezRESUMO
The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.
Assuntos
Animais Geneticamente Modificados/genética , Callithrix/genética , Modelos Animais de Doenças , Células Germinativas/metabolismo , Hereditariedade/genética , Transgenes/genética , Animais , Animais Recém-Nascidos , Callithrix/embriologia , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Transcrição GênicaRESUMO
Severely immunocompromised NOD/Shi-scid IL2Rg (null) (NOG) mice, which show higher engraftment efficiency, are useful as recipients in xenotransplantation studies. We generated a NOG-enhanced green fluorescent protein (EGFP) transgenic (Tg) mouse (NOG-EGFP) that was introduced the EGFP transgene from the C57BL/6-EGFP Tg mouse using the speed congenic method with a marker-assisted selection protocol (MASP). With this method, the selection of the male with the closest NOG strain type was repeated four times. When human cord blood CD34(+) cells were transplanted into NOD/Shi-scid, NOG, and NOG-EGFP mice (N(6)), the engraftment efficiency of the NOG-EGFP mice was significantly higher than that of the NOD/Shi- scid mice and was similar to that of the NOG mice. These results suggest that the NOG-EGFP mice, which were generated using the congenic method with MASP, acquired the immunological properties of the NOG mice.
