RESUMO
The development potential of transgenic adult cells after nuclear transfer (NT) was evaluated. Primary ovine granulosa cells (GC(S)) from a slaughter ovary were transfected with pEGFP-N1 plasmid DNA. Three G418-resistance cell lines (A2, B2 and B4) were used as donor cells in NT. A total of 162 NT blastocysts were then frozen with ethylene glycol solution and stored for five months before transplanted into recipients. Twenty-nine frozen thawed NT blastocysts were transferred into 15 synchronized recipients. Twin lambs (6.9%) derived from B2 line were delivered by cesarean section on day 143 but died after birth. A tumor consisting of lung tissues was found on the surface of left lung of the 4-kg lamb and histological analysis indicated that it resembles a hamartoma. DNA analysis confirmed that two lambs were genetically identical to B2 donor cells. Gene insertion and expression have been detected in fibroblasts cells derived from muscle tissues of the lambs. This study indicates that granulosa cell is a suitable cell type for producing transgenic animals by nuclear transfer. Offspring were produced after long-term storage of NT blastocysts.
Assuntos
Animais Geneticamente Modificados , Clonagem de Organismos/métodos , Células da Granulosa/metabolismo , Animais , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Células Clonais , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Hamartoma/diagnóstico , Neoplasias Pulmonares/diagnóstico , Repetições de Microssatélites , Ovário/metabolismo , Ovinos , Manejo de Espécimes , Fatores de Tempo , TransfecçãoRESUMO
Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti-bull sperm IgG was produced by immunizing rabbits with acrosome-reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome-reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti-bull sperm IgG reacted with sp18 antigens in acrosome-reacted bull sperm head and bull seminal plasma. Anti-bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti-sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti-sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p < 0.001).
