Generation of two-cell cloned embryos from mouse faecal cell.

Cloning animals using nuclear transfer (NT) provides the opportunity to preserve endangered species. However, there are risks associated with the collection of donor cells from a body, which may cause accidental death of the animal. Here, we tried to collect faeces-derived cells and examined the usability of those nuclei as a donor for NT. A relatively large number of cells could be collected from GFP-Tg mouse faeces by this method. After NT, only 4.2% of the reconstructed oocytes formed pseudo-pronucleus. This rate increased up to 25% when GFP and Hoechst were used as a marker to select better cells. However, the reconstructed oocytes/embryos showed several abnormalities, such as shrunken nuclear membranes and abnormal distribution of tubulin, and none of them developed beyond one-cell stage embryos. These developmental failures were caused by not only toxic substances derived from faeces but also intrinsic DNA damage of donor cell nuclei. However, when the serial NT was performed, some of the cloned embryos could develop to the two-cell stage. This method may remove toxic substances and enhance DNA repair in the oocyte cytoplasm. Thus, these results indicate that faeces cells might be useful for the conservation of endangered species when technical improvements are achieved.

Production of cloned mice using oocytes derived from ICR-outbred strain.

Recently, it has become possible to generate cloned mice using a somatic cell nucleus derived from not only F1 strains but also inbred strains. However, to date, all cloned mice have been generated using F1 mouse oocytes as the recipient cytoplasm. Here, we attempted to generate cloned mice from oocytes derived from the ICR-outbred mouse strain. Cumulus cell nuclei derived from BDF1 and ICR mouse strains were injected into enucleated oocytes of both strains to create four groups. Subsequently, the quality and developmental potential of the cloned embryos were examined. ICR oocytes were more susceptible to damage associated with nuclear injection than BDF1 oocytes, but their activation rate and several epigenetic markers of reconstructed cloned oocytes/embryos were similar to those of BDF1 oocytes. When cloned embryos were cultured for up to 4 days, those derived from ICR oocytes demonstrated a significantly decreased rate of development to the blastocyst stage, irrespective of the nuclear donor mouse strain. However, when cloned embryos derived from ICR oocytes were transferred to female recipients at the two-cell stage, healthy cloned offspring were obtained at a success rate similar to that using BDF1 oocytes. The ICR mouse strain is very popular for biological research and less expensive to establish than most other strains. Thus, the results of this study should promote the study of nuclear reprogramming not only by reducing the cost of experiments but also by allowing us to study the effect of oocyte cytoplasm by comparing it between strains.

Birth of cloned mice from vaginal smear cells after somatic cell nuclear transfer.

Less invasive methods for donor cell collection will facilitate reproduction of wild animals using somatic-cell nuclear transfer. Stages of the estrous cycle in mice have long been studies using somatic cells that can be collected from vaginal walls using cotton tipped swabs in a relatively non-invasive manner. In this study, we examined the feasibility of these cells as sources of nuclei for somatic-cell cloning using nuclear transfer. Estrous cycles generally comprise proestrus, estrus, metestrus, and diestrus stages. In the present experiments, more than 60% of cells were nucleated in vaginal smears from all but the estrus stage. However, after somatic-cell nuclear transfer of cells from proestrus, metestrus, and diestrus stages, 66%, 50%, and 72% of cloned embryos developed to the morula/blastocyst, and cloned female mouse birth rates after embryo transfer were 1.5%, 0.3%, and 1%, respectively. These results show that noninvasively collected vaginal smears contain somatic cells that can be used to clone female mice.