Replication of somatic micronuclei in bovine enucleated oocytes

• Autores: Canel, Natalia Gabriela; Bevacqua, Romina Jimena; Hiriart, María Inés; Salamone, Daniel Felipe
• Año de publicación: 2012
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Cell Division; Vol.7; 7:23; http://www.biomedcentral.com/
• Materia: CHROMOSOMES; MICRONUCLEI; OOCYTE; TRANSGENE; BOVINAE
• Institución: Universidad de Buenos Aires. Facultad de Agronomía

Fil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Fil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Animal. Laboratorio de Biotecnología Animal. Buenos Aires, Argentina.
Background: Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.Methods: Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 ug/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 ug/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed (Micronucleus- injected (+) or not (Micronucleus- injected (-) ) to a transgene (50 ng/ul pCX-EGFP) during 5 min. Enucleated oocytes (Enucleated (+) and parthenogenetic (Parthenogenetic (+) ) controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/ul pCX-EGFP. A non-injected parthenogenetic control (Parthenogenetic (-) was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 uM ionomycin for 4 min plus 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (-), Parthenogenetic (-) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher's exact test (p less or equal to 0.05). Results: All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.Conclusions: We have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.