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Myeloid Leukaemia Factor 1 and Haemopoiesis: Aspects of Mlf1's

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Myeloid Leukaemia Factor 1 and Haemopoiesis: Aspects of Mlf1's Myeloid Leukaemia Factor 1 and haemopoiesis: Aspects of Mlf1’s regulation and function Clare Morgan BA BSc (Hons) This thesis is presented for the degree of Doctor of Philosophy September 2010 School of Medicine & Pharmacology, University of Western Australia Centre for Medical Research, Western Australian Institute for Medical Research Table of Contents Abstract _____________________________________________________________ i Declaration _________________________________________________________ iii Statement of Candidate Contribution ____________________________________ iv Publications__________________________________________________________v Acknowledgements __________________________________________________vii List of Non-Standard Abbreviations ____________________________________ viii 1. Literature Review__________________________________________________1 1.1 Haemopoiesis _______________________________________________ 1 1.1.1 Primitive and definitive haemopoiesis___________________________ 1 1.1.2 Haemopoietic stem cells_____________________________________ 3 1.1.3 Purification of murine HSCs by fluorescence-activated cell sorting ____ 4 1.1.4 Lineage commitment and plasticity_____________________________ 7 1.1.5 Leukaemia and cancer stem cells ____________________________ 11 1.2 Myeloid Leukaemia Factor 1 __________________________________ 13 1.3 MicroRNAs_________________________________________________ 20 1.3.1 microRNAs in haemopoiesis and leukaemia ____________________ 25 1.3.2 miRNA-29 family__________________________________________ 26 1.3.3 miRNA-181 family_________________________________________ 29 1.4 Project aims________________________________________________ 31 2 Materials & Methods_______________________________________________33 2.1 Animal biology______________________________________________ 33 2.1.1 Isolation of bone marrow cells _______________________________ 33 2.1.2 Collection of tissue samples _________________________________ 33 2.1.3 Collection and analysis of blood samples_______________________ 34 2.1.4 Competitive bone marrow transplants _________________________ 34 2.2 Cell biology ________________________________________________ 34 2.2.1 Mammalian cell culture_____________________________________ 34 2.2.2 Cryopreservation of cells ___________________________________ 36 2.2.3 Cytocentrifugation of cells___________________________________ 36 2.2.4 Mouse colony-forming cell assays ____________________________ 36 2.2.5 Induction of M1 cells_______________________________________ 38 2.2.6 Transfection of adherent cell lines ____________________________ 38 2.2.7 Amphotropic infection of M1 cells _____________________________ 39 2.2.8 Ecotropic infection of EL4 cells_______________________________ 39 2.3 Cell sorting and flow cytometry________________________________ 39 2.3.1 Flow cytometry ___________________________________________ 39 2.3.2 Magnetic cell separation____________________________________ 41 2.3.3 Fluorescence-assisted cell sorting (FACS)______________________ 42 2.4 Molecular Biology ___________________________________________ 42 2.4.1 Isolation of genomic DNA ___________________________________ 42 2.4.2 Isolation of total RNA ______________________________________ 42 2.4.3 DNase treatment__________________________________________ 43 2.4.4 cDNA synthesis __________________________________________ 44 2.4.5 Quantitative RT-PCR ______________________________________ 44 2.4.6 Polymerase chain reaction __________________________________ 47 2.4.7 Site-directed mutagenesis __________________________________ 49 2.4.8 Restriction enzyme digestion ________________________________ 49 2.4.9 Cloning _________________________________________________ 49 2.4.10 Sequencing______________________________________________ 51 2.4.11 Dual luciferase reporter assay _______________________________ 51 2.4.12 Polyacrylamide gel electrophoresis (PAGE) and Western blotting____ 52 2.4.13 Microarray expression profiling and analysis ____________________ 53 2.5 Statistical analysis __________________________________________ 53 3 Mlf1 expression has lineage specific effects___________________________55 3.1 Introduction ________________________________________________ 55 3.2 Results ____________________________________________________58 3.2.1 Mlf1 effects monoblastoid gene expression _____________________58 3.2.2 Sca-1 is up-regulated in the presence of Mlf1 in M1 cells___________62 3.2.3 Transient knockdown of Mlf1 ________________________________67 3.2.4 Persistent knockdown of Mlf1 in M1 cells _______________________67 3.2.5 Mlf1 suppression does not effect M1 differentiation _______________72 3.2.6 Mlf1 effects T lymphoid gene expression _______________________75 3.2.7 Mlf1 effects T lymphoid cell morphology and viability ______________80 3.3 Discussion _________________________________________________83 4 Mlf1 is regulated by microRNA-29___________________________________ 89 4.1 Introduction ________________________________________________89 4.2 Results ____________________________________________________91 4.2.1 Mlf1 3’ UTR is a predicted target of miRNAs ____________________91 4.2.2 Mlf1 3’ UTR is a target of miR-29, but not miR-181 _______________94 4.2.3 miR-29 down-regulates Mlf1 mRNA and protein levels ____________94 4.2.4 Relationship between miR-29 and Mlf1 expression in haemopoietic cell lines____________________________________________________98 4.2.5 miR-29 expression is inversely correlated with Mlf1 expression during myeloid differentiation _____________________________________102 4.3 Discussion ________________________________________________104 5 Characterisation of Mlf1 transgenic mice____________________________ 107 5.1 Introduction _______________________________________________107 5.2 Results: ank-Mlf1 mice ______________________________________109 5.2.1 Development of ank-Mlf1 transgenic mice _____________________109 5.2.2 Gross phenotypic observations______________________________109 5.2.3 Erythropoiesis ___________________________________________112 5.3 Results: vav-Mlf1 mice ______________________________________121 5.3.1 Development of vav-Mlf1 transgenic mice _____________________121 5.3.2 Gross phenotypic observations______________________________121 5.3.3 Erythropoiesis ___________________________________________124 5.3.4 Lymphopoiesis __________________________________________124 5.3.5 Haemopoietic stem cells and progenitors ______________________128 5.4 Discussion ________________________________________________133 6 General Discussion _____________________________________________ 139 7 References_____________________________________________________ 147 Index of Figures Figure 1.1 The classic model of haemopoiesis. Figure 1.2 The structures of MLF1, NPM and the fusion gene, NPM-MLF1 Figure 1.3 Mlf1 over-expression causes profound morphological changes in J2E cells Figure 1.4 The biogenesis and regulatory mechanisms of miRNAs Figure 1.5 Sequence alignment of microRNAs from the miR-29 and miR-181 families Figure 3.1 qRT-PCR confirms differential expression of transcripts identified by microarray in M1-Mlf1 cells Figure 3.2 Sca-1 is up-regulated in M1-Mlf1 cells Figure 3.3 Over-expression of Mlf1 does not effect Sca-1 expression in other lineages Figure 3.4 Sca-1 is up-regulated during differentiation of M1 monoblastoid cells Figure 3.5 Sca-1 levels are elevated in the presence of exogenous Mlf1 during M1 maturation Figure 3.6 siRNA targeting Mlf1 mRNA Figure 3.7 Transient siRNA knockdown of EGFP-Mlf1 Figure 3.8 Transient siRNA knockdown of endogenous Mlf1 Figure 3.9 Schematic representation of pSR-Mlf1 Figure 3.10 Mlf1 expression in M1-pSR.Mlf1 clonal cell lines Figure 3.11 Suppression of Mlf1 alters Sca-1 expression during M1 differentiation Figure 3.12 Levels of miR-29 expression in haemopoietic cell lines Figure 3.13 Mlf1 over-expression in EL4 cells Figure 3.14 Over-expression of Mlf1 alters EL4 morphology Figure 3.15 Mlf1 over-expression increases apoptosis in EL4 cells in normal and stress conditions Figure 4.1 Predicted miRNA target sites within Mlf1 3’ UTR Figure 4.2 Schematic representations of firefly luciferase reporter constructs Figure 4.3 Mlf1 3’UTR is a direct target of miR-29 miRNAs Figure 4.4 Mlf1 3’UTR is not a direct target of miR-181 Figure 4.5 Mlf1 mRNA expression is reduced by miR-29b, but not miR-181a Figure 4.6 Mlf1 protein expression is reduced by miR-29b Figure 4.7 Levels of miR-29 expression in haemopoietic cell lines Figure 4.8 Mlf1 mRNA expression is inversely correlated to miR-29 levels during M1 differentiation Figure 5.1 Generation of ank-Mlf1 transgenic mice Figure 5.2 ank-Mlf1 bone marrow Figure 5.3 ank-Mlf1 affects foetal development Figure 5.4 ank-Mlf1 affects embryo survival and foetal liver cellularity Figure 5.5 Flow cytometric analysis of ank-Mlf1 erythroid cells Figure 5.6 ank-Mlf1 affects foetal erythropoiesis Figure 5.7 ank-Mlf1 embryos have increased numbers of erythroid-committed progenitors Figure 5.8 Generation of vav-Mlf1 transgenic mice Figure 5.9 Megaloblasts are present in vav-Mlf1 bone marrow Figure 5.10 Flow cytometric analysis of vav-Mlf1 erythroid cells Figure 5.11 B lymphocyte populations of vav-Mlf1 mice Figure 5.12 T lymphocyte populations of vav-Mlf1 mice Figure 5.13 Colony-forming progenitors in vav-Mlf1 bone marrow Figure 5.14 Flow cytometric analysis of vav-Mlf1 haemopoietic stem cells Index of Tables Table 2.1 Cell lines used during this project Table 2.2 Antibodies used in flow cytometry and cell sorting Table 2.3
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