Replication of somatic micronuclei in bovine enucleated oocytes
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 oocyt...
| Otros Autores: | , , , |
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| Formato: | Artículo |
| Lenguaje: | Inglés |
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| Acceso en línea: | http://ri.agro.uba.ar/files/download/articulo/2012Canel.pdf LINK AL EDITOR |
| Aporte de: | Registro referencial: Solicitar el recurso aquí |
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| 022 | |a 1747-1028 | ||
| 024 | |a 10.1186/1747-1028-7-23 | ||
| 040 | |a AR-BaUFA |c AR-BaUFA | ||
| 245 | 1 | 0 | |a Replication of somatic micronuclei in bovine enucleated oocytes |
| 520 | |a 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. | ||
| 653 | 0 | |a CHROMOSOMES | |
| 653 | 0 | |a MICRONUCLEI | |
| 653 | 0 | |a OOCYTE | |
| 653 | 0 | |a TRANSGENE | |
| 653 | 0 | |a BOVINAE | |
| 700 | 1 | |9 38039 |a Canel, Natalia Gabriela | |
| 700 | 1 | |9 67357 |a Bevacqua, Romina Jimena | |
| 700 | 1 | |9 37270 |a Hiriart, María Inés | |
| 700 | 1 | |9 61021 |a Salamone, Daniel Felipe | |
| 773 | |t Cell Division |g Vol.7 (2012), p.2-10 | ||
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| 856 | |u http://www.biomedcentral.com/ |x MIGRADOS2018 |z LINK AL EDITOR | ||
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| 900 | |a 17471028 | ||
| 900 | |a 10.1186/1747-1028-7-23 | ||
| 900 | |a ^tReplication of somatic micronuclei in bovine enucleated oocytes | ||
| 900 | |a ^aCanel^bN. | ||
| 900 | |a ^aBevacqua^bR. | ||
| 900 | |a ^aHiriart^bM.I. | ||
| 900 | |a ^aSalamone^bD. | ||
| 900 | |a ^aCanel^bN. | ||
| 900 | |a ^aBevacqua^bR. J. | ||
| 900 | |a ^aHiriart^bM. I. | ||
| 900 | |a ^aSalamone^bD. F. | ||
| 900 | |a ^aCanel^bN.^tLaboratorio BiotecnologÃa Animal, Departamento de Producción Animal, Facultad AgronomÃa, Universidad de Buenos Aires, Av. San MartÃn 4453, C1417DSE, Buenos Aires, Argentina | ||
| 900 | |a ^aBevacqua^bR. | ||
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| 900 | |a 7:23 | ||
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| 900 | |a Vol. 7 | ||
| 900 | |a CHROMOSOMES | ||
| 900 | |a MICRONUCLEI | ||
| 900 | |a OOCYTE | ||
| 900 | |a TRANSGENE | ||
| 900 | |a BOVINAE | ||
| 900 | |a 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. | ||
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