Factors relevant and possibly limiting to the development of somatic cell clones include procedure, type of donor nucleus, and recipient oocyte. Preparation of the ooplast for nuclear transfer involves a procedure that removes chromosomes and, inadvertently, the meiotic spindle and surrounding cytoplasm. This results in elimination of the oocyte genome, but is presumably associated with the loss of factors beneficial for the development of the clone after introduction of the somatic cell nucleus. Preliminary findings indicate that oocyte chromosome removal after brief treatment with microtubule depolymerizing drugs improves mouse clone development. We will therefore examine in detail, the disassembly and assembly of the spindle under the influence of microtubule inhibitors in both murine and bovine oocytes to determine the kinetics of spindle disassembly during chromosome removal. Optimal parameters for spindle retention during chromosome removal will be assessed for developmental consequences. Identifying the timing of such events will provide the basis for future studies to identify what factors associated with the spindle are developmentally beneficial for somatic cell clones. Aside from the recipient ooplasm, properties of the somatic donor nucleus influence development apparent in the development and phenotypes of clones from different cell types. It unknown if differences exist between donor nuclei of the same population, allowing the development of a small number of clones. This should be reflected in the developmental diversity of clones derived from the same cell type. However, the early developmental demise of the majority of somatic cell clones precludes a full examination. Our preliminary data indicate that development of clones can be extended into later development by aggregation with other clones or normal embryos. The low total cell number of blastocyst stage clones also suggests that clone failure may be related to a proliferation defect early in development.
In Specific Aim 2 we will determine the extent of developmental contribution potential by producing chimeras of clones with normal embryos. The number clones that can contribute to preimplantation and fetal development will be ascertained, as well as the extent and tissue-type of contribution. Both assessment of clone contribution in chimeras and the relevance of spindle-associated factors will provide novel basic information about reprogramming of somatic cell nuclei.
| Eckardt, Sigrid; Leu, N Adrian; Kurosaka, Satoshi et al. (2005) Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres. Reproduction 129:547-56 |
| Kehler, James; Tolkunova, Elena; Koschorz, Birgit et al. (2004) Oct4 is required for primordial germ cell survival. EMBO Rep 5:1078-83 |
| Kurosaka, Satoshi; Eckardt, Sigrid; McLaughlin, K John (2004) Pluripotent lineage definition in bovine embryos by Oct4 transcript localization. Biol Reprod 71:1578-82 |
