Construction of eukaryotic vector of small hairpin interfering RNA against NYD-SP5 / 中华男科学杂志

ABSTRACT

<p><b>OBJECTIVE</b>NYD-SP5 is a newly cloned gene highly expressed in human testes, which consists of 3 598 nucleotides including a 1 027-amino acid open reading frame. It is a human-mouse homologous gene. The domain analysis indicated that the NYD-SP5 protein is a transmembrane protein. This study aimed to design and establish recombinant plasmids of small hairpin interfering RNA (shRNA) against NYD-SP5, and to pave the way for the analysis of the function of NYD-SP5 in the testis using the transgenic mouse model.</p><p><b>METHODS</b>Four sequences of oligonucleotides with the small hairpin structure were designed based on the NYD-SP5 mRNA sequence. Recombinant plasmids were constructed by cloning these oligonucleotides into pGPU6/GFP/Neo vectors. Interfering plasmids against GAPDH were established as positive controls and those targeting non-specific genes used as negative controls. The positive constructs were verified by enzyme digestion and sequencing.</p><p><b>RESULTS</b>Plasmid screening and sequencing showed the sequences of the recombinant plasmids to be the same as the shRNA transcribed sequences, which indicated the successful establishment of the recombinant vectors.</p><p><b>CONCLUSION</b>The shRNA expression vector targeting NYD-SP5 could be established successfully.</p>