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 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.
| Main Authors: | , , , |
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| Format: | Texto biblioteca |
| Language: | eng |
| Subjects: | CHROMOSOMES, MICRONUCLEI, OOCYTE, TRANSGENE, BOVINAE, |
| Online Access: | http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber=46834 |
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