Title

Nuclear reprogramming and culture requirement for somatic cell nuclear transfer in cattle and mice

Date of Completion

January 2007

Keywords

Biology, Molecular|Biology, Cell|Biology, Physiology

Degree

Ph.D.

Abstract

The present study aims to investigate the nuclear reprogramming and culture requirement for somatic cell nuclear transfer (SCNT) in Cattle and Mice. Mammalian cloning has been reported in a dozen of mammalian species that resulted in the live clones; however, cloning efficiency by SCNT is still very low. A number of factors have been reported to affect the cloning efficiency. In order to address these questions, in first study was designed to optimize in vitro culture condition of bovine embryos. A beneficial effect of sodium citrate and Sigma-BSA was found on the development of bovine IVF embryos in CR1aa medium co-cultured with somatic cells. Bovine embryos produced in serum free CR1aa medium showed a competent blastocyst development with morphological and cytological features indistinguishable from those derived from serum-containing medium. Further experiments, using embryos cultured in a somatic-cell free system, revealed an unknown factor necessary for embryonic post-compaction development. This factor is also present in FBS and BSA from Sigma possibly as an unremoved containment while it is likely removed in ICPbio-BSA by chromatographical fractionation and related purifications. ^ Secondly, our nuclear transfer experiments demonstrated that premature chromosome condensation (PCC) is not an indispensable prerequisite for the competent reprogramming of a differentiated somatic genome in cattle. The direct exposure of the donor nucleus to the MII cytoplast, presumably containing high levels of MPF, for a relatively short period of time, is sufficient to trigger a cascade of nuclear reprogramming and developmental events. Higher efficiency of blastocyst development was obtained by immediate activation. Similar in vivo developmental potentials of NT embryos derived from substantially varied protocols (IA vs. DA), and the birth of live clones from both treatments, suggest that cattle may represent a unique species with a greater plasticity available for mechanical, biochemical, and physiological manipulation. ^ Finally, to address the fundamental question-whether the cloned animals are derived from adult stem cell, we tested the hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells (HSCs), progenitor cells (HPCs), and granulocytes. We found that the cloning efficiency increases directly with the state of differentiation in the hematopoietic differentiation hierarchy system and that direct SCNT of terminally differentiated post-mitotic granulocytes leads to term development of cloned pups. Our study suggests that somatic cell animal clones, such as Dolly, could, indeed, be derived from differentiated somatic cells. The results clearly demonstrate that there is no apparent advantage in using primarily isolated hematopoietic stem and progenitor cells over differentiated cells as nuclear donors with the current SCNT-based cloning technology. ^