Replication of somatic micronuclei in bovine enucleated oocytes

• Main Authors: Canel Natalia, Bevacqua Romina, Hiriart María Inés, Salamone Daniel
• Format: Article
• Language: English
• Published: BMC 2012-11-01
• Series: Cell Division
• Subjects: Micronuclei; Oocyte; Chromosomes; Transgene
• Online Access: http://www.celldiv.com/content/7/1/23

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. <it>In vitro</it> 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/μl 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/μl 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 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and <it>egfp</it> expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−), Parthenogenetic (−) and <it>in vitro</it> fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05).</p> <p>Results</p> <p>All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed <it>egfp</it> 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.</p> <p>Conclusions</p> <p>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.</p>