Expression Of NYD-SP5 Gene In Mouse Testis And Establishment Of NYD-SP5 Interference Vector

ObjectiveNYD-SP5 is a newly found gene that is highly expressed in human testis~[1].It consists of 3598 nucleotides,including the 1027 amino acid open reading frame.Gene sequence analysis revealed that NYD-SP5 had high homology with mouse testis AK019535 gene and its encoded protein BAB31783.This indicates that the gene NYD-SP5 is a human-mouse homologous gene.The domain analysis of NYD-SP5 gene indicates that its protein is a transmembrane protein.In this study,methods of immunohistochemistry and in situ hybridization were used to study the expression and location of NYD-SP5 in mouse testis.In addition, recombinant plasmids of small hairpin interfering RNA(shRNA) against NYD-SP5 were designed and established.These results laid solid foundation for analyzing functions of NYD-SP5 in the testis by using transgenic mouse model.METHODSThe expression and localization of NYD-SP5 protein in mouse testis tissue was studied with immunohistochemical method;Expression and localization of NYD-SP5 mRNA was studied with in situ hybridization.Four NYD-SP5 pGPU6/GFP-shRNA vectors were constructed.Recombinant plasmid was constructed based on NYD-SP5 mRNA sequence.Four pairs of oligonucleotides were synthesized and inserted into plasmid pGPU6/GFP to generate shRNA eukaryotic expression vectors.Interfering plasmid from GAPDH were established as positive control and interfering plasmid targeting none gene served as negative control.The recombinant vectors were verified with enzyme digestion and sequencing.RESULTSIn situ hybridization showed that there was brown stain in spermatogenic cells in the cytoplasm in mice spermatogenic tubule in the experimental group,whereas the spermatogenic cells in the control group were not stained,suggesting that NYD-SP5 mRNA exist in spermatogenic cells.Immunohistochemical analysis showed that mouse spermatogenic cells of the tubule seminiferous epithelium and lumen ciliated cells were stained brown,but the cells the control group were not stained.This suggested that NYD-SP5 protein exist in spermatogenic cells in spermatogenic tubule in mouse and the cilia in the lumen.Enzyme digestion and electrophoresis analysis and gene sequencing analysis of the inserted nucleotide sequences confirmed that targeted sequences had been successfully inserted into the the expected site.The recombinant plasmid sequence was the same as the shRNA transcribed sequence indicating that the recombinant vectors had been successfully established.ConclusionNYD-SP5 mRNA and protein were expressed in mouse germ cells,suggesting that the gene may play a role in spermatogenesis.The shRNA expression vector targeting human NYD-SP5 gene was successfully established.This study has laid the foundation for the further study,i.e.to construct an animal model of transgenic mice expressing NYD-SP5 gene and study the function of NYD-SP5.