Date of Award:

5-2007

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Kenneth L. White

Committee

Kenneth L. White

Committee

Thomas D. Bunch

Committee

John D. Morrey

Committee

Quinton A. Winger

Committee

Daryll B. DeWald

Committee

Mark C. Healey

Abstract

Since the production of the first sheep by somatic cell nuclear transfer a great deal of effort has been made to improve efficiency and to understand nuclear reprogramming mechanisms. Unfortunately efficiency remains low, and nuclear reprogramming mechanisms remain uncharacterized. The objectives of this research were to identify factors associated with somatic cell nuclear transfer efficiency and to analyze the transcriptome of blastocyst-stage clone and control embryos and cotyledonary tissue in an effort to elucidate mechanisms responsible for the low developmental efficiency and high post-implantation losses.

The experiments reported here identify factors including oocyte source and timing of activation following nuclear transfer that yield improved efficiencies. It was determined the use of cow oocytes for somatic cell nuclear transfer results in improved in vitro development and increased pregnancy rates. These data further indicate prolonged exposure of the donor nucleus to pre-activated oocyte cytoplasm results in increased nuclear fragmentation and reduced developmental efficiency in vitro.

Several aberrantly expressed genes were identified in nuclear transfer blastocysts and cotyledons that could impact cloning efficiency. Major histocompatibility complex I and down-regulator of transcription 1 were overexpressed in nuclear transfer blastocysts, and retinol binding protein 1 was overexpressed in nuclear transfer cotyledons. The functions of these genes in immune response, transcriptional regulation, and retinol binding and transport make them attractive candidates for further nuclear transfer research.

Expression levels of six developmentally important genes were analyzed in various stages of preimplantation nuclear transfer embryos by real-time polymerase chain reaction to determine the timing of nuclear reprogramming following nuclear transfer. Five of the six genes were aberrantly expressed multiple developmental stages, however by the blastocyst stage only one gene was aberrantly expressed. These data indicate reprogramming is delayed in nuclear transfer embryos resulting in over- or under-expression of developmentally important genes during early embryogenesis.

These experiments report factors associated with improved nuclear transfer efficiency; provide insight into potential mechanisms for low developmental rates, abnormal placentation, and fetal loss of clones; and characterize the timing of nuclear reprogramming following somatic cell nuclear transfer.

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