Title page
CONTENTS
ABBREVIATIONS 9
ABSTRACT 10
1. INTRODUCTION 11
2. MATERIALS AND METHODS 16
2.1 Database Sequence Retrieval 16
2.2 Selection Criteria for HSC EST Libraries 17
2.3 Cloning of Murine P2Y10 17
2.4 Cell Culture and Cell Lines 18
2.5 RT-PCR 18
2.6 Retroviral Vectors, Production, Transduction and Concentration 19
2.7 Flow Cytometry Analysis and Cell Sorting 20
2.8 Cytospin 21
2.9 Cell Proliferation Assay 21
2.10 Cell Cycle Analysis 22
3. RESULTS 23
3.1 Unigene/EST Gene Index Screen 23
3.2 Characterization of Murine P2Y10 Receptor 26
3.3 Expression of Murine P2Y10 receptor in HSCs 28
3.4 Expression of Murine P2Y10 in CD4, CD8 cell in thymocyte and CD3, B 220 cells in spleenocyte 28
3.5 Retroviral Introduction of Murine P2Y10 Receptor into Ba/F3 cell line 32
3.6 P2Y10 Overexpression Inhibits Proliferation for Ba/F3 cells 35
3.7 P2Y10 Gene Regulates Cell Cycle in Ba/F3 cells 35
4. DISCUSSION 39
REFERENCE 43
SUMMERY IN KOREAN 50
Table 1. Numbers of mouse HSC specific Unigene/EST in dbESTs and Unigenes 25
Fig 1. Deduced Amino Acid Sequence of the mP2Y10 Proteins and Its Alignment with Other Homologous Receptor Sequences using t-coffee. The putative membrane-spanning domains were designated with bar above the sequence. (GenBank Accession Number. Human: NP_055314, Mouse: NP_766023, Rat: NP_446093.1). 27
Fig 2. P2Y10 Expression in the Hematopoietic Stem/Progenitor Cells of bone marrow. Sorted K+S+L-, K+S-L-, K-S+L- and K-S-Lcells were isolated from the mouse BM (A) and RT-PCR expression analysis of mP2Y10 was shown (B). 30
Fig 3. RT-PCR analysis of mP2Y10 in sorted T and B lymphocyte from thymus and spleen. Each fraction of T and B lymphocytes was isolated from thymus and spleen and then conducted the mP2Y10 expression by RT-PCR (A) was indicated the result of analysis with T thmocytes (A) and (B) from splenocytes. 31
Fig 4. Schematic Representation of the MSCV and mP2Y10 Retroviruses and Transient Transfection into Retroviral Package Cell Line 33
Fig 5. Retroviral Introduction and Gene Expression of P2Y10 into Ba/F 3 Cell Line 34
Fig 6. Retardation of cell proliferation by mP2Y10 overexpression. mP2Y10 transduced Ba/F3 cells were cultured for indicated days and determined the cell proliferation by MTT. 37
Fig 7. Cell Cycle Analysis of P2Y10 Transduced Ba/F3 cell line. 38