Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

Summer 2020

Thesis Director

Gary A. Bulla

Thesis Committee Member

Thomas Canam

Thesis Committee Member

Antony O. Oluoch

Abstract

In mammals, a complex system of regulatory signals distinguishes tissues, structures and functions. Combinations of transcription factors and co-factors regulate activation and repression of genes that result in cellular differentiation. Whole genome arrays allow the monitoring of genomic expression in specific tissues. Fibroblast microarray studies have shown candidate genes that may be involved in fibroblast identification, including genes that express transcription factors Prrx1, Snai2 and Twist1. A previous study showed that the Prrx1 and Snai2 could reactivate a fibroblast phenotype in hybrid cells that had lost fibroblast identity. Furthermore, overexpression of these factors in liver-derived cells strongly repressed liver gene expression and activated fibroblast expression. Based on these observations, expression plasmids containing Prrx1, Snai2 and Twist1, expression cassettes were transfected independently into mouse Neuro2A neuronal cells using standard transfection technique, followed by the selection of G418-resistant clones (pool and clones). Expression of essential fibroblast marker genes and neuronal genes was monitored in transfected cells and non-transfected cells using qualitative real-time polymerase chain reaction (RT-qPCR) on cDNA derived for isolated RNA. Results showed that, surprisingly, little activation of expression occurred for any of the fibroblast genes tested. Rather, strong repression of several fibroblast genes was observed. However, both Snai2 and Prrx1 did appear to strongly repress several neural genes tested, suggesting a partial reprograming of the Neuro2A cells away from a neural phenotype.

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