[Construction of eukaryotic vector of small hairpin interfering RNA against NYD-SP5]

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.

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.

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.

The shRNA expression vector targeting NYD-SP5 could be established successfully.