利用家畜、禽的選性繁殖技術，生產期望性別的後裔，長久以來一直為畜牧業者所努力與期待。經由雄性性別特異性基因JUMONJI, AT-rich interactive domain 1D (RBP2-like) (JARID1D)的選殖與操作，利用免疫方法，以製備性別特異性抗體的方式分離豬與牛攜帶X與Y染色體精子是本研究最終的目標。當雄性的小鼠個體皮膚移植到同血緣的雌性個體上時，JARID1D 能製造出一個H-Y抗原來產生免疫反應。豬與乳牛之JARID1D基因cDNA估計在3.5~4.5 Kb之間，而目前基因已知的片段分別只有457 bp及99 bp。在本實驗中，選殖豬與牛JARID1D的方法是運用生物資訊學 (bioinformatics)與聚合酶鏈反應 (polymerase chain reaction, PCR)以及建立cDNA library與篩選法。我們使用人類JARID1D基因當探針或模板來尋找NCBI及TIGR資料庫中與人類JARID1D基因相似之豬與牛的expressed sequence tags (ESTs)，結合兩資料庫的結果並用CAP3軟體來做組合(assembly)。我們建立了豬與牛的cDNA library，並搜尋了豬的cDNA library。為了在小鼠上測試此模式，合成了mouse Jarid1d H-Y epitope胜肽片段, TENSGKDI對兔子進行免疫，來製備小鼠anti-Jarid1d H-Y epitope TENSGKDI抗體。點墨法(dot blotting)顯示了抗體對mouse Jarid1d H-Y epitope, TENSGKDI的專一性，但是西方點墨法並未顯示相同結果。免疫螢光法(immunofluorescence analysis)的結果顯示小鼠精子與該抗體的結合並沒有1:1的比例關係，顯示抗體的專一性仍待探討。實驗結果顯示部分豬的JARID1D基因已經成功選殖，並嘗試製備小鼠性別特異性抗體，但其抗體對小鼠精子表面蛋白質表現之特異性需再進一步確認。

Sex pre-selection in livestock offspring to produce dairy cows and specific male and females lines for heterosis in swine are important economic goals in animal husbandry. The long-term goal of the study is to separate porcine and bovine chromosome X- or Y-bearing sperms using sex-specific antibodies. H-Y antigen encoded by jumonji, AT-rich interactive domain 1d (Rbp2-like) (Jarid1d) is defined as a male histocompatibility antigen that causes rejection of male skin grafts by female recipients of the same inbred strain of rodents. Porcine and bovine JARID1D genes are estimated between 3.5 kb and 4.5 kb, however there was only 457 bp and 99 bp respectively found in the database. In this study, the porcine and bovine sex-specific genes, JARID1D were cloned using in silico cloning, PCR and cDNA library screening. We used human JARID1D gene as a probe or template to search the ESTs of porcine and bovine in NCBI and TIGR databases and combined the results from two databases and assembled the ESTs using CAP3 program. We also constructed porcine and bovine testis cDNA libraries, and performed porcine testis cDNA libraries screening. To study this model in the mouse, Jarid1d H-Y epitope TENSGKDI was synthesized and used to immune the rabbits. Dot blotting analysis revealed the specificity of the immuned antibodies to mouse Jarid1d H-Y epitope TENSGKDI, but western blotting analysis did not confirm the results. Immunofluorescence analysis of mouse sperms reacted with anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibody didn’t show a 1:1 ratio of stained sperms to unstained sperms. The specificity of this antibody needed to be improved. The data shows partial JARID1D gene of pig was cloned, and the anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies were prepared, however the specificity of anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies to surface protein expression on mouse sperms needs more confirmations.

CONTENTS
Chinese abstract ---------------------------------------------------------------------------1
English abstract ---------------------------------------------------------------------------2
Chapter 1. Literature review------------------------------------------------------------3
Chapter 2. In silico and PCR-based cloning of porcine and bovine Y-chromosome specific gene jumonji, AT rich interactive domain 1d (RBP2-like) (JARID1D)
2.1 Introduction --------------------------------------------------------------------------14
2.2 Materials -----------------------------------------------------------------------------14
2.3 Methods ------------------------------------------------------------------------15
2.4 Results -------------------------------------------------------------------------------17
2.5 Discussion ---------------------------------------------------------------------------20
Tables -----------------------------------------------------------------------------------22
Figures ----------------------------------------------------------------------------------47
Chapter 3. Construction of porcine and bovine testis cDNA libraries and screening of JARID1D gene on a porcine testis cDNA library
3.1 Introduction --------------------------------------------------------------------------52
3.2 Materials and methods ---------------------------------------------------------------52
3.3 Results ------------------------------------------------------------------------------57
3.4 Discussion ---------------------------------------------------------------------------58
Tables -----------------------------------------------------------------------------------60
Figures ----------------------------------------------------------------------------------61
Chapter 4. Preparation of anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies and test the availability in a mouse model
4.1 Introduction -------------------------------------------------------------------------65
4.2 Materials and Methods----------------------------------------------------------------65
4.3 Results ------------------------------------------------------------------------------71
4.4 Discussion --------------------------------------------------------------------------73
Tables -----------------------------------------------------------------------------------75
Figures ---------------------------------------------------------------------------------76
References --------------------------------------------------------------------------------80

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