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2-2 Agarose Gel Electrophoresis Image of PCR Products DEVELOPMENT OF A FLOXED ADIPONECTIN TRANSGENIC MOUSE MODEL USING CRISPR-CAS9 TECHNOLOGY By PETER ELIAS DIB A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017 © 2017 Peter Elias Dib To Elias & Bader Dib ACKNOWLEDGMENTS I would like to express my utmost gratefulness for the assistance and direction that my mentor, Dr. Sergei Zolotukhin, has provided me throughout the past year and a half. I thank my committee members, Dr. Edgardo Rodriguez and Dr. James Resnick for their valuable input on my project during every meeting. I thank Ruan Oliviera from Dr. Maurice Swanson’s laboratory for his helpful advice on the CRISPR-Cas9 protocol. I thank Dr. Damien Marsic, Dr. Olecksander Kondratov, and Sean Crosson from Dr. Sergei Zolotukhin’s laboratory for their constant support and help inside and outside of the laboratory. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 6 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 INTRODUCTION .................................................................................................... 11 Overview of Obesity and Adiponectin ..................................................................... 11 Transgenic Adiponectin Mice .................................................................................. 13 Cre-Lox System ...................................................................................................... 14 CRISPR-Cas9 ......................................................................................................... 16 2 CONSTRUCTION OF FLOXED ADIPONECTIN GENE ......................................... 23 APN Exon 2 and Homologous Arms ....................................................................... 23 Repair Template DNA for CRISPR-Cas9 ................................................................ 23 3 DESIGNING AND TESTING GUIDES FOR CRISPR-CAS9 .................................. 30 Designing Guide RNA ............................................................................................. 30 Testing Guide RNA Efficiency ................................................................................. 32 4 CONCLUSIONS ..................................................................................................... 36 5 MATERIALS AND METHODS ................................................................................ 38 PCR Amplification of Exon 2 and Homologous Arms ............................................. 38 Gibson Assembly of Construct ................................................................................ 39 Bacterial Transformation & Colony Screening ........................................................ 40 Guide RNA Design and Oligonucleotide Orders ..................................................... 40 Plasmid Construction and Bacterial Transformation ............................................... 41 Transfection ............................................................................................................ 42 Surveyor Assay ....................................................................................................... 43 LIST OF REFERENCES ............................................................................................... 45 BIOGRAPHICAL SKETCH ............................................................................................ 48 5 LIST OF TABLES Table page Table 3-1. Guide ssDNA Sequence Table .................................................................... 34 6 LIST OF FIGURES Figure page 1-1 Cre-Lox experimental design .............................................................................. 19 1-2 CRISPR-Cas9 .................................................................................................... 20 1-3 NHEJ vs HR. ...................................................................................................... 21 2-1 Agarose Gel Electrophoresis Images. ................................................................ 26 2-2 Agarose Gel Electrophoresis Image of PCR products. ....................................... 27 2-3 Repair Template DNA Assembly ........................................................................ 28 2-4 1% Agarose Gel Image of Restriction Digest of 14 Clones. ............................... 29 3-1 Surveyor Nuclease Assay Image. ....................................................................... 35 7 LIST OF ABBREVIATIONS AAV Adeno-associated Virus APN Adiponectin ATP Adenosine Tri-Phosphate bp Base pair CreER Cre recombinases CreERT Tamoxifen-dependent Cre recombinase CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats- Associated Cas9 Endonuclease D Loop Displacement Loop DMEM Dulbecco’s Modified Eagle Medium ES Embryonic Stem FBS Fetal Bovine Serum gRNA Guide RNA HR Homologous Recombination LB Lysogeny Broth mRNA Messenger RNA PAM Protospacer Adjacent Motif PBS Phosphate-Buffered Saline PCR Polymerase Chain Reaction PNK T4 Polynucleotide Kinase PYY Peptide Tyrosine-Tyrosine tracrRNA Trans-activating crRNA TS Target Sequence YR Neuropeptide Y Receptor ZFN Zinc Finger Nuclease 8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF A FLOXED ADIPONECTIN TRANSGENIC MOUSE MODEL USING CRISPR-CAS9 TECHNOLOGY By Peter Elias Dib May 2017 Chair: Sergei Zolotukhin Major: Medical Sciences Obesity is a major health concern facing the world population which raises the risk for a number of severe associated health impediments such as cardiovascular diseases, diabetes, respiratory disorders, renal dysfunction, hepatic dysfunction, sleep disorders and cancer. One of the protein hormones that has been linked to obesity is adiponectin (APN) which is produced and secreted by adipose tissue, an endocrine organ, that has long been linked to obesity. APN’s functions include modulation of energy metabolism along with insulin sensitivity and fatty acid oxidation. APN secretion is disrupted due to fat buildup in obesity, leading to the onset of obesity-linked diseases. APN is present in the oral cavity but its function isn’t very well understood yet in the context of it being a salivary component. APN transgenic mouse models have been studied to further understand the function and effects of adiponectin. Gene-targeted knockout is a technique that entails mutating a specific gene in vitro and then replacing the normal copy in the genome with a mutant form. Studies in an inducible APN Cre-Lox knockout mouse model is needed to help improve the understanding of APN and its effects on whole body metabolism while having pharmacological control over the timing 9 of gene expression. Development of this mouse model benefits from CRISPR/Cas9 system of RNA guided genome editing which has revolutionized genetics research. The core purpose of this project is to set up and test in vitro all the essential components for the development of a floxed APN transgenic mouse. APN exon 2 holds the initiation codon of the APN messenger RNA (mRNA) transcript which is translated by the ribosome into APN protein. Flanking APN exon 2 by two unidirectional LoxP sequences will lead in the excision and degradation of APN exon 2 from the genomic DNA as a result of the action of Cre recombinase of the Cre-Lox system, disrupting the gene and stopping protein production. This project benefits heavily from the molecular biology techniques that have been well established in the past along with newer and more cutting edge methods such as CRISPR-Cas9 and Cre-Lox genome editing systems. 10 CHAPTER 1 INTRODUCTION Overview of Obesity and Adiponectin Obesity is gaining recognition as a primary health threat that hinders nearly 37% of the US population’s quality of life according to the most recent statistics done by the Center for Disease Control (CDC). It is a complex metabolic disorder mostly influenced by environmental and genetic factors (Wright et al., 2012). It often leads to the development of severe comorbidities such as diabetes, cardiovascular disease, and cancer. Obesity is a topic on which many interpretations are strongly held in the lack of scientific support to back up those analyses (Casazza et al., 2015). For that reason and many others, obesity has been on the rise and finding a suitable therapy is highly imperative. Gastrointestinal (GI) peptides and hormones have been proven to influence appetite, satiation, and energy expenditure through interactions with cognate receptors expressed in the brain or in peripheral nerve fibers making them the focus of many studies aimed at understanding obesity and its causes. One of those studies focused on adiponectin (APN), a physiologically active polypeptide hormone secreted by adipose tissue that displays insulin-sensitizing, anti-inflammatory, and antiatherogenic properties (Shklyaev et al., 2003). APN is also one of the
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