Construction Of CDNA Expression Library And Isolation Of Tubule Spermatogenic Cells From Adult Seminiferous Tubule

Objective:1. To construct cDNA plasmid expression library from adult seminiferous tubule,2. To isolate tubule spermatogenic cells.Method: 1. Total RNA was prepared with Trizol from adult seminiferous tubule, and then mRNA was further isolated and purified by Oligo(dT) cellulose powder. The mRNA was reverse-transcribed into ds-cDNA, and then ds-cDNA was cloned into expression plasmid vector pSPORT ? and transformed into DH5α competent cells with high efficient electroporation. Finally, cDNA expression library was constructed. Afterwards, two interested ESTs were analyzed. 2. Spermatogenic cells were isolated from adult seminiferous tubule by means of modified discontinuous Percoll gradient (22﹪, 30﹪, 35﹪, 40﹪) centrifugation, combined with optimized enzymatic digestion.Result:1. The unamplified cDNA library contained about 1.1×106 cfu,the percentage of recombinant clones was about 93%,and the average size of the insert was about 1.3kb;2. One ESTs was 31% of amino acid sequence homologous with gonadotropin-releasing hormone receptor Π,while the other possessed 26% of amino acid sequence homologous with calmodulin. The later ESTs contained an ORF (opening reading frame), which coded 128 amino acids and a putative conserved domains , was 60.9% in amino acid homology with calmodulin and other correlated protein family domains;3. Most of spermids (91.7%) and immature spermatogenic cells (88.6%) were enriched at 22% fraction and 30% fraction respectively, where the highest cell density appeared at 30% fraction. Conclusion: 1. cDNA expression library from adult seminiferous tubule was constructed sucessfully;2. One EST may represent homologous protein sequence of human gonadotropin-releasing hormone receptorΠ,and the other may be the homologous protein of calmodulin;3. Spermids and immature spermatogenic cells were enriched with high purification.