UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ The Visualization, Quantification and Modeling of Genomic Instability in the Mouse and in Cultured Cells A dissertation submitted to the graduate school of the University of Cincinnati In partial fulfillment of the requirements for the degree of Doctorate of Philosophy (PhD) In the department of Molecular Genetics, Biochemistry and Microbiology of the College of Medicine 2006 by Jon Scott Larson B.S., University of Toledo, 1999 Committee: Dr. James Stringer (chair) Dr. Tom Doetschman Dr. Joanna Groden Dr. Carolyn Price Dr. Peter Stambrook Abstract Multicellular organisms are mosaic in nature because of genetic alterations that occur in somatic cells. There are many factors that can contribute to the formation of such alterations including aberrant DNA repair, environmental insults, epigenetic modification, errors in DNA replication and errors in chromosome duplication/segregation. To further the study of the distributions, frequencies and rates at which some alterations can occur, mouse reporter models were implemented. The Tg(βA-G11PLAP) transgenic mutation reporter mouse harbors an allele (G11 PLAP ) that is rendered incapable of producing its functional enzyme because of a reading frame shift caused by an insertion of 11 G:C basepairs. Spontaneous deletion of one G:C basepair from this mononucleotide repeat restores gene function, and cells with PLAP activity can be detected histochemically. G11 PLAP mice enable mutant cells to be visualized in situ and were used to study variation during early development, in the germline, under oxidative stress and in solid tumors. To study LOH in diverse cell types in the body another reporter model was implemented. Mice that carry two different fluorescent protein genes as alleles of a locus were generated to address this issue because LOH would change a cell’s phenotype from bichrome to monochrome. As a step in assessing the utility of this approach, we derived MEF and ES cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. FACS showed that the vast majority of cells in each line expressed the 1 two marker proteins at similar levels, but populations exhibited extrinsic and intrinsic noise with respect to expression. In addition, cells with a monochrome phenotype were frequent (10-4). In ES cells, all monochrome events were accompanied by allele loss. Mitotic recombination appeared to be the major cause, although UPD also appeared to have contributed to LOH. These cells provided a novel assay for studying genetic/karyotypic stability of cultured ES cells. Results obtained from studies with these cells support the need for caution regarding the use of cultured stem cells in therapy. 2 Acknowledgements There have been a few people who have impacted my life in such a way that it forever changed who I am and helped to shape me into the person that I am today. I owe credit to these invaluable influences for the encouragement, direction and determination given to me for everything leading up to the preparation of this document: dissertation for the Ph.D. degree granted by the Department of Molecular Genetics, Biochemistry and Microbiology at the University of Cincinnati, College of Medicine. Thank you to my Ph.D. advisor/ mentor, Dr. Jim Stringer. I sometimes wonder how I was so fortunate to get a project in his lab. He has provided more guidance and support towards me than I thought I would need, giving me the much need reality checks that kept me on track. I have learned much from him. I would also like to thank the Stringer family; Saudra, Hillary and Alice. They have been like family to me, forgiving my occasional blunders (sorry Mr. Wiggles et. al.). They will always be in my heart and mind. Jim and the past/present members of the lab (Saundra Stringer Ph.D., Scott Keely Ph.D., Megan Hersh Ph.D., Jared Fischer and Carolyn Tindal) have all shared both the joys and frustrations of my successes, as well as the utter failures. I thank you for many hours of intellectually stimulating, entertaining and sometimes absolutely absurd conversions, and interlaboratory activities. I will sincerely miss working with all of you. I would also like to thank my graduate committee members, Tom Doetschman Ph.D., Joanna Groden Ph.D., Carolyn Price Ph.D. and Peter 3 Stambrook Ph.D. I couldn’t imagine having a more enthusiastic and supportive group of mentors, all renowned in their areas of specialties. They were instrumental in training me, always pushing me to dive deeper, think clearly, and work harder. “Thank you” to the members of the department who have extended to me both their assistance and moral support, throughout my tenure as a graduate student. To Rachel Sellmeyer, Dorie Lane, Peggy Casselman , Vicki Morris, Felicia Romaine, Moying Yin, Tina Grisham, Issac Houston, Brock Schwitzer, Justin Huddleson, Kelly Flory, Elizabeth Loreux and Jorge Muniz, my most sincere gratitude. You all have become dear friends whom I will miss. Lastly, and most importantly, I would like to thank the love of my life Iva Dostanic (Larson). We met and shared many great times while in ‘Molgen’. She has been my muse and support through the most challenging, emotional and exciting times. Thanks Bebe! 4 5 Table of Contents List of Abbreviations p8 Chapter 1 – Introduction Abstract p12 Background p13 Chapter 2 – Modeling Variation in Tumors in vivo Abstract p26 Introduction p27 Results p30 Discussion p42 Materials and Methods p45 Chapter 3 - Increased Mutation in Mice Genetically Predisposed to Oxidative Damage in the Brain Abstract p50 Introduction p51 Results p53 Discussion p59 Materials and Methods p60 Chapter 4 - Impact of Mismatch Repair Deficiency on Genomic Stability in the Maternal Germline and during Early Embryonic 6 Development Abstract p63 Introduction p64 Results p68 Discussion p74 Materials and Methods p80 Chapter 5 - Expression and Loss of Alleles in Cultured Mouse Embryonic Fibroblasts and Stem Cells Carrying Allelic Fluorescent Protein Genes Abstract p85 Introduction p86 Results and Discussion p89 Conclusion p103 Materials and Methods p108 Chapter 6 – Dissertation Summary p119 References p123 7 Abbreviations Aif apoptosis inducing factor APAF-1 apoptotic protease activating factor 1 APC adenomatous polyposis coli APE1 Apurinic endonuclease 1 APRT adenine phosphoribosyltransferase BAX BCL2 associated X BCIP 5-bromo-4-chloro-3-indolphosphate BCL2 B-cell lymphoma 2 Bl6 black six mouse strain Bp basepair(s) CFP cyan fluorescent protein Chk1 checkpoint kinase1 c-myb Cellular DNA binding proteins encoded by the myb gene DMEM Dulbecco’s modified eagle media DNA deoxyribonucleic acid DNA-PKcs Protein Kinase, DNA activated, catalytic subunit dPBS Dulbecco’s phosphate buffered saline DSB double strand break E. coli Escherichia coli E2F-4 transcription factor that control expression of a variety of genes involved in cell cycle regulation 8 EMS Ethanomethylsulfate ES cell embryonic stem cell FACS fluorescent activated cell sorting FISH fluorescent in situ hybridization Flash FLice-ASsociated Huge protein FVB/N friend virus B/NIH mouse strain G11 PLAP reporter transgene that contains a mononucleotide run of eleven GC base pairs in the human PLAP gene hMSH3 human MutS homolog 3 hMSH6 human MutS homolog 6 HNPCC Hereditary nonpolyposis colorectal cancer Hq Harlequin ICE Family of aspartate-specific cysteine proteases, caspases IGF2R insulin-like growth factor II receptor IR ionizing radiation LIF Leukemia inhibitory factor LOH loss of heterozygosity MAPK mitogen activated protein kinase MEF mouse embryonic fibroblast MgCl2 magnesium chloride MLSN1 Melastatin 1 9 MMR mismatch repair MMTV mouse mammary tumor virus MR mitotic recombination MSI microsatellite instability Myc family of retrovirus-associated DNA sequences (myc) originally isolated from an avian myelocytomatosis virus NADH reduced nicotinamide adenine dinucleotide (NAD+) Neu neu Proto-Oncogene Protein, A cell surface protein- tyrosine kinase receptor that is found to be overexpressed in a significant number of adenocarcinomas PBS phosphate buffered saline PCR polymerase chain reaction Pdcd8 programmed cell death 8 PLAP placental alkaline Phosphatase PMS2 post meiotic segregation 2 PyMT polyoma virus middle T-antigen RAD50 human homologue of a yeast gene, required for spontaneous and induced mitotic recombination, meiotic recombination and mating-type switching. RAS family of viral oncogenes Sky spectral karyotyping 10 SV40 simian virus 40 Taq Thermos aquaticus TCF family of DNA-binding proteins that are primarily expressed in T-LYMPHOCYTES Tg(βA-G11PLAP) transgenic mouse with a G11 PLAP allele driven by a human beta actin promoter TGFbRII transforming growth factor beta receptor II UPD uniparental disomy XPG xeroderma pigmentosum YFP yellow fluorescent protein 11 Chapter 1 Introduction Abstract Multicellular organisms are mosaic in nature because of genetic alterations that occur in somatic cells. There are many factors that can contribute to the formation of such alterations including aberrant DNA repair, aberrant cell cycle, environmental insults (e.g. oxidative stress, radiation), epigenetic