DIFFERENTIAL GENE AND PROTEIN EXPRESSION IN PIG NUCLEAR TRANSFER EXTRAEMBRYONIC MEMBRANES AND BLASTOCYST STAGE EMBRYOS A Dissertation presented to the Faculty of the Graduate School University of Missouri In Partial Fulfillment Of the Requirement for the Degree Doctorate of Philosophy by KRISTIN MARIE WHITWORTH Dr. RANDALL S. PRATHER, Dissertation Supervisor DECEMBER 2010 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled GENE AND PROTEIN EXPRESSION IN PIG NUCLEAR TRANSFER EXTRAEMBRYONIC MEMBRANES AND BLASTOCYST STAGE EMBRYOS Presented by Kristin M. Whitworth A candidate for the degree of Doctor of Philosophy And hereby certify that in their opinion it is worth of acceptance. Professor Randall S. Prather Professor Rodney D. Geisert Professor Peter Sutovsky Professor R. Michael Roberts Professor Yuksel Agca ACKNOWLEDEMENTS I would like to thank the members of my committee Dr. Randall Prather, Dr. Peter Sutovsky, Dr. Rodney Geisert, Dr. R. Michael Roberts and Dr. Yuksel Agca for all of their guidance and suggestions throughout my PhD program. I couldn‟t have asked for a better committee, always prompt and helpful. My advisor, Dr. Prather has been so patient with me as I have worked through this degree and I appreciate this so much. There are also so many members of the Prather lab that I would like to thank. This project involved collection of samples from over 150 pigs and Lee Spate assisted with every pig. His help and friendship will always be appreciated along with August Rieke who was always by our side. Thank you Dr. Clifton Murphy for performing all the embryo transfers needed to complete this project and for helping me appreciate our country and those who have sacrificed for it. Thank you for all the close friendships that have been developed while working in the lab. A fun job with great people has made this process so enjoyable. Thank you Jason Ross, Clay Isom, Jeff Whyte, Melissa Samuel, Bethany Bauer, Eric Walters, Kyle Dobbs, Mingtao Zhao, Jiude Mao, Jennifer Teson and Lisa Tracy, I have enjoyed working with all of you. I‟d also like to thank Jianguo Zhao for all of his advice and help with the Scriptaid experiments. Thank you to Dr. Peter Sutovsky and his lab members Miriam Sutovsky and Gauri Manandhar for helping with this project. The microscopy work from from Gauri and Miriam greatly aided in the publication of Chapter 2. Also thank ii you for the help of Dr. Jonathan Green‟s lab and his senior research specialist, Tina Egen. Their help with Western Blotting, Immunohistochemistry and expertise in placental biology was greatly appreciated. I would also like to thank my supportive family. My husband Jon spent many evenings and weekends caring for our children, Erin, Jordan and Kathleen, so that I could study and write. I couldn‟t have done it without him. My parents, Bill and Kathy Whitworth, have spent the last four years taking care of the kiddos during Christmas and summers breaks so that I wouldn‟t have to take time off from my project. Thank you Jon, thank you Mom and Dad and thank you kids for being patient. I hope that the three of you will grow up to value education. Lastly I would like to thank my friends that continued their education “later in life” and inspired me to do the same. iii TABLE OF CONTENTS AKNOWELEDGEMENTS……………………………………………………………………….ii LIST OF FIGURES………………………………………………………………………………x LIST OF TABLES………………………………………………………………………………xiii NOMENCLATURE……………………………………………………………………………..xv ABSTRACT…………………………………………………………………………………….xvii Chapter 1. REVIEW OF LITERATURE…………………………………………………………….1 Introduction……………………………………………………………………………....1 Brief History of Nuclear Transfer in Animals………………………………………....2 The Role of Enhanced Green Fluorescent Protein (EGFP) in NT Pigs…………...4 Nuclear Transfer and Genetically Modified Pigs for Use in Agriculture….….…….6 Nuclear Transfer and Genetically Modified Pigs for Use as Human Disease Models……………………………………………………………….8 Nuclear Transfer and its Effects on Placentation…………………………………..13 Pig………………………………………………………………………………13 Mouse………………………………………………………………………….14 Cow…………………………………………………………………………….18 Why is important to study porcine placentation and nuclear transfer?.....19 Nuclear Reprogramming…………………………………………………………...…20 Fusion and Activation After Nuclear Transfer………………………………………21 Oocyte Activation……………………………………………………………..21 Activation and Fusion of NT Zygotes……………………………………….22 Effects of Nuclear Transfer on Gene Expression…………………………………..23 iv Pig………………………………………………………………………………25 Cow……………………………………………………………………………..29 Histone Deacetylase Inhibitors and How They Improve Nuclear Transfer………32 Histone Deacetylation and Acetylation……………………………………..32 Description of the Histone Deacetylase Inhibitor, Scriptaid………………34 Using HDACi to Improve Nuclear Transfer……………….……………………...…36 Pig………………………………………………………………………………36 Cow……………………………………………………………………………..39 Mouse…………………………………………………………………………..41 Other Methods to Improve Nuclear Transfer……………………………………….45 Altering DNA Methylation…………………………………………………….45 Inhibition of Proteasomal Machinery………………………………………..45 Conclusions…………………………………………………………………………….47 2. Method of Oocyte Activation Affects Cloning Efficiency in Pig……………………50 Abstract...……………………………………………………………………………….50 Introduction……………………………………………………………………………..52 Materials and Methods………………………………………………………………..55 Animal Use………………………………………………………………….…55 Chemicals……………………………………………………………………...55 Preparation of pCAGG-EGFP Construct………………………………...…55 Preparation of Donor Cells…………………………………………………..56 Transfection of Donor Cells with pCAGG-EGFP………………………….57 Preparation of Recipient Oocytes and NT…………………………………57 Fusion and Activation of the Oocyte and Donor Cell……………………..58 Embryo Transfer and Embryo Collection…………………………………..59 v Pregnancy Rates and Recovery Rates……………………………………..60 Nuclei Count in Blastocyst-stage Embryos………………………………...61 Immunofluorescence………………………………………………………….61 Results………………………………………………………………………….………61 Fusion Rates…………………………………………………………………..61 Donor Cell Nuclear Remodeling…………………………………………….62 Mean Number of Nuclei per Blastocyst…………………………………….63 Pregnancy Rates and Recovery Rates…………………………………….63 Birth of pCAGG-EGFP Positive Piglets…………………………………….65 Discussion………………………………………………………………………………65 Conclusions…………………………………………………………………………….69 Acknowledgements……………………………………………………………………69 3. Activation Method Does Not Alter Abnormal Gene Expression and Development in Cloned Pigs…………………………………………………………78 Abstract…………………………………………………………………………………78 Introduction……………………………………………………………………………..80 Materials and Methods………………………………………………………………..83 Ethical Guidelines……………………………………………………………..83 MIAME Compliance…………………………………………………………..84 Chemicals……………………………………………………………………...84 Animals, Donor Cell Line and Oocytes……………………………………..84 Embryo Transfer………………………………………………………………86 Sample Collection for Microarray, Real-time PCR and Western Blotting Analysis………………………………………………86 vi RNA Isolation and cDNA Synthesis…………………………………………87 Sex Determination of Extraembryonic Membranes……………………….87 Microarray Reference Design Experiment…………………………………88 Microarray Analysis by Genespring 7.3.1………………………………….89 Database for Annotation, Visualization and Integrated Discovery (DAVID)…………………………………………………………………....…..90 Real-time PCR Validation……………………………………………………91 Western Blotting and Densitometry…………………………………………92 Measurement of Acid Peptidase Activity (APA)……………………………94 Immunohistochemistry…………………………………………………….….94 Image Acquisition ……………………………………………………….……96 Results………………………………………………………………………………….97 Supplemental Files……………………………………………………………97 Pregnancy Rates……………………………………………………………...97 Sex Determinations of Extraembryonic Membranes……………………...98 Labeling Efficiency……………………………………………………………98 Microarray Comparisons……………………………………………………..98 Database for Annotation, Visualization and Integrated Discovery (DAVID)……………………………………………………………………….100 Real-Time PCR Validation………………………………………………….100 Western Blotting and Densitometry………………………………………..101 Acid Peptidase Activity……………………………………………………...102 Immunohistochemisty……………………………………………………….102 Discussion…………………………………………………………………………….104 Conclusion…………………………………………………………………………….110 vii Acknowledgements…………………………………………………………………..110 4. Scriptaid Corrects Gene Expression of Several Aberrantly Reprogrammed Transcripts in Nuclear Transfer Pig Blastocyst Stage Embryos………………...118 Abstract………………………………………………………………………………..118 Introduction……………………………………………………………………………120 Materials and Methods………………………………………………………………123 Supplemental Files………………………………………………………….123 Ethical Guidelines……………………………………………………………124 MIAME Compliance…………………………………………………………124 Chemicals…………………………………………………………………….124 Animals, Donor Cell Line and Oocytes……………………………………124 Treatment Group Description: BLIVV, BLIVF, NT1, NT2, NT3, NTS…125 Embryo Transfer……………………………………………………………..126 Nuclear Protein Extraction and HDAC Assay…………………………….127 Quantification of Global Methylation in NT Embryos…………………….127 Sample Collection for Microarray and Real-time PCR…………………..128 RNA Isolation and cDNA Synthesis of the Reference Sample………....129 RNA Isolation and Amplification of the Embryo and Cells………………130 Microarray Reference Design Experiment………………………………..130 Labeling Efficiency…………………………………………………………..131 Microarray Analysis by Genespring 7.3.1…………………………………131 Identification of Aberrantly Reprogrammed Transcripts…………………132 Resequencing of ESTs from the 5′ Direction……………………………..133 Database for Annotation, Visualization and Integrated Discovery (DAVID)……………………………………………………………………….133 viii Real-time