Cloning of Sheep tnp2 Gene and Transcription Analysis of Round Spermatid Cells in the in vitro Co-culture System

The transition protein-2 gene (tnp2) have been reported as the round spermatid-specific marker gene. However, the DNA sequence of tnp2 gene in Ovis aries have not been reported. In order to study the sheep round spermatid-specific marker gene, we designed primers according to the reported Bos taurus tnp2 gene cDNA sequences in the GenBank. The partial CDS was amplified by RT-PCR. The PCR fragment was inserted into the T-A cloning vector pMD19-T. The partial nucleotide sequences of Mongolia sheep tnp2 gene were compared with the counterpart sequences of Bos taurus, and the nucleotide homology was 95.3%. Using the primers designed according to the tnp2 cDNA sequence, the tnp2 gene transcription expression in the co-cultured cells derived from four-months old sheep testis was assessed by RT-PCR. The results demonstrate that the round spermatid cells are generated in the co-culture system until ten weeks in vitro. The cDNA cloning and sequencing lay down a foundation for further study on spermatogenesis of sheep.