比格犬ERβ剪切异构体ERβ419沉默表达载体的构建及筛选

Construction and identification of ERβ419 gene silencing recombined vectors of beagle ERβ isoforms

目的构建最有效的慢病毒介导RNAi干扰序列,未来将应用于比格犬ERβ419的未知生物学功能的探索。方法模拟比格犬ERβ419靶细胞筛选针对目的基因mRNA设计的最佳干扰序列实验,通过qRT-PCR和Western Blot技术筛选最佳沉默表达载体序列。结果qRT-PCR结果显示,ERβ419-shRNA1(P<0.01)和ERβ419-shRNA3(P<0.01)的差异表达极显著,Western Blot结果与qRT-PCR结果一致,ERβ419-shRNA3的干扰效果最佳。结论 ERβ419-shRNA3最有效地抑制了目的基因的表达,未来将应用于探索比格犬ERβ419未知生物学功能对比格犬生殖系统影响的研究,并进而预防和治疗比格犬繁殖机能障碍性疾病。

Objective To construct and identify retroviral-mediated short hairpin RNA （shRNA） expression vectors of ERβ419, and explore ERβ419 unknown biological function in beagles in future. Methods To screen out the most effective gene silencing sequence of beagle ERβ419 mRNA using qRT-PCR and Western Blot assays, imitate beagle estrogen target cells. Results qRT-PCR results showed, ERβ419-shRNA1 （P 〈 0. 01 ） and ERβ419-shRNA3 （ P 〈 0. 01 ）differed significantly, Western Blot result as same as qRT-PCR,ERβ419-shRNA3 is the best choice. Conclusion Beagles ERβ419-shRNA3 retrain most effectively target gene repression. It is applied to explore ERβ419 unknown biological function in beagles reproductive system, and to prevent and treat beagles reproductive function diseases.