Tet1 Function in Intestinal Stem Cell Differentiation Restricted; Files Only

Abstract

Intestinal stem cells (ISCs) located at the bottom of crypts rapidly proliferate and differentiate into intestinal epithelial cells (IECs) that migrate to their designated location where they perform their specific function. The single layer of IECs that lines the intestines plays an important role in intestinal function. Chromatin modifying enzymes influence the expression of genes and the differentiation of stem cells. Previous work in our lab has identified ten-eleven translocase 1 (TET1) as a regulator of ISC differentiation, and studies have shown its influence on early development of the intestines. However, the impact of TET1 on adult intestines has not been identified. In this study, we characterized the phenotypes of an inducible Tet1 knockout mouse model (Tet1iKO). The number of mature IECs was significantly altered, while this phenotype difference was also found to be influenced by environmental factors. To clarify the function of Tet1, we designed a new plasmid OrgOne that allows inducible expression of gene of interest by doxycycline in combination of PiggyBac transposase that inserts the sequence into the host cell genome, to generate Tet1 gain-of-function organoids. The plasmid was tested by cloning GFP into the backbone and the results showed that the plasmid allows inducible expression of the inserted gene of interest.

Table of Contents

Table of Contents

Chapter 1………………………………………………………………………………......……………..1

Tet1iKO Phenotype and Environmental Dependency…………………………………..1

1.1 Introduction…………………………………………………………………………………………..1

1.2 Results ……………………………………………………………………………………......………2

1.3 Discussion and Future Directions …………………………………………………………4

1.4 Figures ……………………………………………………………………………………......………5

1.5 Material and Methods …………………………………………………………………………..8

Chapter 2 ……………………………………………………………………………………....………..10

Plasmid Design for Inducible Overexpression for Organoid Transfection……10

2.1 Introduction …………………………………………………………………………………………10

2.2 Results and Discussions ………………………………………………………………………11

2.3 Future Directions …………………………………………………………………………………13

2.4 Figures …………………………………………………………………………………………………14

2.5 Material and Methods ………………………………………………………………………..…17

References ………………………………………………………………………………………………..20

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, General Biology, Cell Biology, Genetics
Keyword	Intestinal Stem Cells Tet1
Committee Chair / Thesis Advisor	Gracz, Adam, Emory University
Committee Members	Srinivasan, Shanthi, Emory University Deal, Roger, Emory University

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