Laboratorio Biotecnología Animal, Departamento de Producción Animal, Facultad Agronomía, Universidad de Buenos Aires, Av, San Martín 4453, C1417DSE, Buenos Aires, Argentina.
Cell Div. 2012 Nov 22;7(1):23. doi: 10.1186/1747-1028-7-23.
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.
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. 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/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 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≤0.05).
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.
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.
微细胞介导的染色体转移（MMCT）技术被开发出来，用于将少量染色体导入宿主细胞。我们设计了一种将 MMCT 与体细胞核转移相结合的新技术，该技术包括将体细胞核内的微核注入去核卵母细胞，并诱导其细胞机制复制该微核。这将允许分离和操作单个或少量的体染色体。
通过在 0.05μg/ml 秋水仙素中孵育 46 小时，然后用 2mg/ml 丝裂霉素处理 2 小时，从成年牛成纤维细胞中产生微核。最后用 10μg/ml 细胞松弛素 B 处理 1 小时。体外成熟的牛卵母细胞被机械去核，并在细胞质内注射一个以前暴露过（微核注射（+））或未暴露过（微核注射（-））5 分钟转基因（50ng/μl pCX-EGFP）的体细胞核内微核。去核卵母细胞（去核（+））和孤雌激活（孤雌激活（+））对照物被注入含有少于 10pl 50ng/μl pCX-EGFP 的 PVP 的细胞质中。还包括一个非注射的孤雌激活对照（孤雌激活（-））。注射后 2 小时，卵母细胞和重构胚胎通过在 5μM 离子霉素中孵育 4 分钟+1.9mM 6-DMAP 3 小时进行激活。评估卵裂阶段和 egfp 表达。通过 DAPI 染色证实 DNA 复制。第 2 天，对微核注射（-）、孤雌激活（-）和体外受精（IVF）胚胎进行核型分析。通过 Fisher 精确检验（p≤0.05）确定处理之间的差异。
所有实验组均进行了第一次细胞分裂。有趣的是，少数微核注射胚胎表现出 egfp 表达。DAPI 染色证实了大多数评估胚胎中微核的复制。核型分析显示，所有微核注射胚胎的每个卵裂球的染色体数都少于 15 个（1-13 个），而 IVF 和孤雌激活对照物均未显示少于 30 个染色体/展开物。
我们开发了一种新的方法来复制体细胞核内微核，利用卵母细胞的复制机制。这可能是一种用于染色体转移的有用工具，此前可以针对该工具进行转基因操作。
