In Silico, Molecular Cloning and Characterization of Porcine and Bovine JARID1D Genes

• Main Authors: Yuan-jhih Tsai, 蔡淵智
• Other Authors: Yow-ling Shine
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/79415624678676097835

碩士 === 國立中山大學 === 生物醫學科學研究所 === 92 === 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.