Date of Award

5-2016

Degree Type

Honors College Thesis

Department

Chemistry and Biochemistry

First Advisor

Faqing Huang, Ph.D.

Advisor Department

Chemistry and Biochemistry

Abstract

Embryonic stem cells (ESCs) are characterized by their two special properties: pluripotency and self-renewal. Pluripotency is the ability of ESCs to differentiate into any cell type upon expression of specific proteins called transcription factors. In order to induce differentiation, transcription factors specific for the particular cell type have to be introduced and expressed in the ESCs. The introduction of transcription factors can be achieved by using either DNA, RNA, or protein. According to the Central Dogma of Molecular Biology, DNA is first transcribed into messenger RNA (mRNA), which is then translated to make a functional protein molecule. Among the available methods, direct introduction of mRNA is the safest and most efficient approach for achieving the expression of transcription factors. However, the methods in mRNA transfection has to be tested first. First, the MyoD gene was inserted downstream from the T7 promoter in a plasmid. The plasmid was then cloned in E. coli. We then extracted the plasmid DNA and amplified the MyoD gene by polymerase chain reaction (PCR). The amplified MyoD DNA was concentrated and purified by ethanol precipitation. MyoD mRNA was prepared by in vitro transcription catalyzed by T7 RNA polymerase using the recovered MyoD DNA as the template. Finally, the resulting RNA was purified by using 100 kDa molecular weight cutoff spin columns and quantified by UV spectroscopy. This method of RNA transcription had produced 145.16 μg RNA, comparative to the standard 160 μg RNA produced by a longer template. Using this method, modified mRNA can be produced and used to transfect the embryonic stem cells in order to induce differentiation into myocytes.

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