Date of Award

5-2017

Degree Type

Honors College Thesis

Department

Chemistry and Biochemistry

First Advisor

Faqing Huang, Ph.D.

Advisor Department

Chemistry and Biochemistry

Abstract

The development of therapeutic immune responses from the manipulation of embryonic stem cells (ESCs) via induction of synthetic RNA with 5’ capped and 3’ poly(A)tailed ends would lead to development of stem cell therapy. A necessary step in attaining such a goal is to first produce an mRNA transcript from a plasmid containing the open reading frame (ORF) for a transcription factor for cellular activation.

In this research, the DNA plasmid pMD4 encoding MyoD was transcribed into synthetic mRNA. The plasmid was first amplified using Polymerase Chain Reaction (PCR), and analyzed using gel electrophoresis. The amplified template was purified via ethanol precipitation. The template was then transcribed using T7 RNA polymerase and an RNase inhibitor along with RNase free buffers and solutions. Gel filtration Spin Column Chromatography was then used to separate and purify the RNA transcript so that it could be properly quantified using UV-Vis Spectroscopy. A concentration yield of 4.2g/L of the RNA transcript was obtained (Figure 9) and stored for further downstream cellular differentiation and innate immune response application.

Included in

Biochemistry Commons

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