THE FUNCTION OF ABCF1 IN IMMUNITY AND MOUSE DEVELOPMENT by Sara Morgen Wilcox B.Sc, University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2011 © Sara Morgen Wilcox, 2010 ii Abstract ABCF1 is an ABC (ATP binding cassette) transporter protein that lacks trans-membrane domains. Gene expression of ABCF1 has been shown to increase upon tumour necrosis factor alpha (TNFα) stimulation [1]. This is significant as TNFα is a pro-inflammatory cytokine produced by macrophages and T cells and has a number of functions in the immune response [2]. ABCF1 is thought to function in translational initiation by interacting with the eukaryotic initiation factor 2 (eIF2) [3]. We have determined that ABCF1 is an essential gene in development by the generation and characterization of mice that have a gene trap insertion in the ABCF1 gene, which terminates the expression of the ATP binding cassettes. Homozygous ABCF1 knock-out (ABCF1-/-) mice are embryonic lethal at 3.5 days post coitus (dpc) while heterozygous ABCF1 knock-out (ABCF1+/-) mice appear to be developmentally normal. This thesis utilizes the ABCF1 gene trapped mouse model which contains a β-geo (β- galactosidase /neomycin) reporter gene in the ABCF1 gene to examine the endogenous ABCF1 promoter expression. This allows us to concurrently observe the activity of the ABCF1 promoter in all tissues through sectioning and X-Gal staining. This analysis provides further insight into the physiological function of ABCF1 by identifying the tissues and cell types that have the highest levels of promoter activity. Interestingly, ABCF1 appeared to be expressed in the marginal zone of the spleen and areas surrounding the lymphoid follicles. Macrophages, which were isolated from spleens of ABCF1+/- mice, were found to be hyper-responsive to stimulation by TLR ligands, particularly LPS, while macrophages derived from the bone marrow were found to be hypo-responsive. When challenged with LPS, ABCF1+/- mice produced altered cytokine production compared to their wild-type controls. iii Upon challenge with Listeria monocytogenes, ABCF1+/- mice succumbed to infection sooner than their ABCF1+/+ littermates. Taken together, these data indicate that ABCF1 is necessary for survival and development and likely has a role in the regulation of cytokines. Thus ABCF1 may play a significant role in regulating inflammation and pathogen induced “cytokine storm”. iv Preface The XKO97 ES cell was aggregated and injected into C57/B6 blastocysts by Jian Xin (Michael Smith Laboratories, University of British Columbia, Vancouver, BC). Blastocysts were isolated and Clone 1 was cultured by Lonna Munro (Michael Smith Laboratories, University of British Columbia, Vancouver, BC). The day 5.5-7.5 embryos were removed by Dr. Juan Hou (BC Cancer Agency, University of British Columbia, Vancouver, BC) Drs. Hebrit Adissu and Susan Newbigging (Toronto Centre for Phenogenoics, Toronto, Ont) helped analyse structures in the β gal stained tissues in chapter 3. Dr Hebrit Adissu took pictures 4.3 and 4.5 in chapter 4. Rayshad Gopaul (Department of Zoology & Michael Smith Laboratories, University of British Columbia, Vancouver, BC) managed one of the mouse colonies. UBC Animal care approval certificates A07-0270 and A09-0824 were acquired for this research. This thesis was written by Sara Wilcox and edited by Cheryl Pfeifer, Lonna Munro and Wilf Jefferies. v Table of contents Abstract ............................................................................................................................... ii Preface ............................................................................................................................... iv Table of contents ................................................................................................................ v List of tables ...................................................................................................................... ix List of figures ...................................................................................................................... x List of abbreviations ......................................................................................................... xv Acknowledgments ......................................................................................................... xviii Dedication ........................................................................................................................ xix Chapter 1: General introduction ......................................................................................... 1 1.1 Immunology .............................................................................................................. 1 1.1.1 The innate immune system ................................................................................. 1 1.1.2 Innate immunity and macrophages .................................................................... 2 1.1.3 TLRs ................................................................................................................... 3 1.1.4 Inflammation ...................................................................................................... 5 1.1.5 Cytokines ............................................................................................................ 6 1.1.6 Autoimmunity .................................................................................................... 7 1.2 Eukaryotic translation ............................................................................................... 8 1.2.1 Translation control ........................................................................................... 12 1.2.2 Translational control and disease ..................................................................... 14 1.3 ABC transporters..................................................................................................... 15 1.3.1 The ABC family of transport proteins .............................................................. 15 1.3.2 TAP transporters .............................................................................................. 22 1.3.3 ABCF1 ............................................................................................................. 24 1.4 Rationale and hypothesis ........................................................................................ 28 1.5 Thesis synopsis ....................................................................................................... 29 1.6 References ............................................................................................................... 31 Chapter 2: Gene trap disruption of the ABCF1 gene causes lethality at early embryonic stages of development....................................................................................................... 36 2.1 Overview ................................................................................................................. 36 2.2 Introduction ............................................................................................................. 37 vi 2.2.1 Gene trapping ................................................................................................... 38 2.2.2 Mouse embryogenesis ...................................................................................... 40 2.3 Materials and methods ............................................................................................ 44 2.3.1 XK097 and mice ............................................................................................... 44 2.3.2 Collection of mouse blastocysts ....................................................................... 45 2.3.3 Genomic DNA isolation ................................................................................... 45 2.3.4 Genotyping ....................................................................................................... 46 2.3.5 ES cell line derivation .......................................................................................... 47 2.4 Results ..................................................................................................................... 49 2.4.1 The ES cell line XK097 from BayGenomics contains a gene trap vector in the ABCF1 gene. ............................................................................................................. 49 2.4.2 ABCF1 homozygous knock-out mice produced from the XKO97 cell line are embryonic lethal. ....................................................................................................... 54 2.4.3 Clone 1, an ABCF1-/- knock-out ES cell line derived from blastocysts, is viable. ........................................................................................................................ 58 2.5 Discussion ............................................................................................................... 60 2.6 References ............................................................................................................... 63 Chapter 3: The characterization of the ABCF1 promoter activity in tissues and the analysis of the ABCF1-deficient CD8+ T cells. ................................................................ 65 3.1 Overview ................................................................................................................