Title

Study of nuclear reprogramming: DNA replication, therapeutic cloning, and improvements of tetraploid complementation

Date of Completion

January 2010

Keywords

Chemistry, Biochemistry|Health Sciences, Human Development

Degree

Ph.D.

Abstract

Nuclear reprogramming refers to the process of erasing the transcriptional program of somatic cells and initiating that of an embryo. Somatic cell nuclear transfer (SCNT) is the only approach to date to complete the reprogramming process and re-establish the pluripotent status within a few cell divisions. This dissertation was composed of three parts of experiments to elucidate the nuclear reprogramming. ^ Firstly, experiments were to address DNA replication process involving in the reprogramming during the first cell cycle of mouse SCNT embryos. We demonstrated that SCNT embryos revealed distinct patterns in the structure of chromatin loops and revealed 2 hours delay of progression of DNA replication from parthenogenesis embryos. Moreover, obvious alteration could be observed when SCNT embryos underwent treatment of epigenetic modulator, trichostatin A (TSA). Because TSA have been shown the beneficial effect to nuclear reprogrammability, therefore, the efficient reprogramming of mouse cloned embryos might be related to DNA replication and associated with histone remodeling. ^ A major application of the SCNT technology is therapeutic cloning, the generation of histocompatible embryonic stem (ES) cells for tissue replacement purposes. The second objective of the dissertation was to apply low density lipoprotein receptor knockout (Ldlr-/-) mouse as a CVD disease model for therapeutic cloning. The result indicated that Ldlr-/- nuclear transferred embryonic stem (ntES) cells could be derivate from fibroblasts after SCNT. Besides, the Ldlr-/- ntES cells could be maintained and provided for gene knock-in to generate gene-repaired (Ldlr-/-) ntES cells. In vitro differentiation and chimera generation also showed that cells could express pluripotent markers and were capable to differentiate to three germ layers. The potential of target differentiation and cell transplantation of ntES cells are needs more studies prior to further regenerative biology applications. ^ Finally, to verify in vivo developmental potential of the reprogrammed cells unbiasely, experiments were performed to optimize the stringent criterion for pluripotent stem cells: tetraploid complementation assay. We compared various embryo stages for host embryos and new medium recipes by small molecules (PD98059 and pluripotin(SC1)) supplementation. We demonstrated that stem cells were able to incorporate into tetraploid embryos with a wide range from 2-cell to blastocyst stages and fullterm mice can be generated. The efficiency could be restricted when the host embryos were under embryo compaction and polarization. Furthermore, ES cell derivation efficiency and the pluripotency can be improved after cultivating stem cells in the present of PD or SC1 molecules. ^