Journal Article
Construction of interference plasmid targeting caprine PHGPx gene and its inhibitory effect on PHGPx expression in co-cultured spermatogenic cells of goats [2012]
Zhang Chunxiang, Shanxi Agricultural University, Taigu (China), College of Animal Science and Veterinary Medicines; Shi Liguang, Chinese Academy of Tropical Agricultural Sciences, Danzhou (China), Tropical Crop and Livestock Genetic Resources Institute; Wang Qian, Shanxi Agricultural University, Taigu (China), College of Animal Science and Veterinary Medicines;
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The objectives of this study were to construct the specific interference plasmid targeting caprine PHGPx gene and identify the inhibitory effect on PHGPx expression in co-cultured spermtogenic cells of goats. Four siRNAs which had potential effect in interference were designed by using the online software in Ambion Company. Short hairpin siRNAs (shRNAs) were cloned into linearized pGPU6/GFP/Neo vector after annealing treatment siRNA in vitro. The pGPU6/GFP/Neo-PHGPx plasmid was identified by Pst I and BamH I restriction analysis and sequencing. The co-cultured spermtogenic cells were transfected by pGPU6/GFP/Neo-PHGPx with Lipofectamine 2000TM. The inhibitory effect of PHGPx gene was detected by RT-PCR and Western blotting. The four recombinant plasmids, pGPU6/GFP/Neo-PHGPx-150, 182, 214 and 248 were confirmed successfully by sequencing and Pst I and BamH I restriction analysise, then transiently transfected into the co-cultured spermatogenic cells. The PHGPx mRNA level in the co-cultured spermatogenic cells dramatically decreased after transfection by the recombinant plasmids pGPU6/GFP/Neo-PHGPx-150, 214 and 248. The average optical density of western-blotting results showed that recombinant plasmid pGPU6/GFP/Neo-PH-GPx-214 inhibited the expression of PHGPx protein significantly (P0.01). The pGPU6/GFP/Neo-PHGPx-150, 182, 214 and 248 were successfully constructed. PHGPx gene was inhibited by recombinant plasmid pGPU6/GFP/Neo-PHGPx-214, which can be used in
further study for PHGPx function.
