Degree

Doctor of Philosophy (PhD)

Department

Animal Sciences

Document Type

Dissertation

Abstract

The ability to produce genetically superior livestock has established somatic cell nuclear transfer (SCNT) as an invaluable tool in commercial livestock production. Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. Several small molecules have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the “roadblocks” encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and completely reactivate the embryonic stem cell (ESC) genome. If small molecules are used to “prime” the somatic cells to be used as donor cells in SCNT, the efficiency of nuclear reprogramming during SCNT may be enhanced.

We first assessed the effect of pre-treatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast (BFF) cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen activated protein kinase/ERK kinase (MEK) inhibitor; CHIR99021, a glycogen synthase kinase-3 (GSK3) inhibitor; and NuP0178, a G9a histone methyltransferase inhibitor. No significant difference in transcript levels for Oct-4, Nanog, or Sox-2 was detected, indicating that this combination of small molecule inhibitors does not have an effect on the expression of Oct-4, Nanog, and Sox-2 in BFF cells.

We next sought to assess the effects this combination of small molecule inhibitors has on the epigenetic state of Oct-4, Nanog, and Sox-2 in BFF cells. Chromatin Immunoprecipitation was used to quantify the enrichment of key histone modifications on the promoter regions of Oct-4, Nanog, and Sox-3 in BFF cells treated with and without PD0325901, CHIR99021, and NuP0178 over time. Time, treatment, and a time*treatment interaction were found to have a significant effect on the histone modifications analyzed. Determining how the expression of these factors alters the epigenetic marks in the promoter regions of key pluripotency-associated genes will allow for the development of defined conditions which best mimic the epigenetic landscape of ESC, ultimately leading to engineering the ideal donor cell for successful SCNT.


Date

1-17-2019

Committee Chair

Bondioli, Kenneth

DOI

10.31390/gradschool_dissertations.4803

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