Key Laboratory of Animal Biotechnology of National Ministry of Agriculture, Institute of Veterinary Immunology,and Research Laboratory of Virology, Immunology & Bioinformatics, Division of Veterinary Microbiology & Virology, Department of Preventative Medicine, College of Veterinary Medicine,and Investigation Group of Molecular Virology, Immunology, Oncology & System Biology, Center for Bioinformatics, Northwest A&F University, Yangling 712100, Shaanxi, China; Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, State Key Laboratory of Veterinary Biotechnology, Harbin, Heilongjiang, China.
Res Vet Sci. 2013 Dec;95(3):1006-11. doi: 10.1016/j.rvsc.2013.08.013. Epub 2013 Aug 25.
To extend the use of RNAi in chicken, we have developed a RNA interference (RNAi) system using a shortened chicken 7SK (ch7SK) promoter. The results stated that the cloned ch7SK promoter includes multiple Oct-1 motifs, SPH domain, PSE and TATA box, without CACCC box. All RNAi groups driven by ch7SK promoter showed significant mean fluorescence intensity (MFI) reduction. In the pch7SK-shEGFP transfected DF-EGFP cell culture, the MFI reduction ratio was smaller than the pmU6-shEGFP did. In the pmU6-shEGFP transfected Vero-EGFP cell culture, the MFI was reduced significantly than the pch7SK-shEGFP did. In summary, the essential part of ch7SK promoter was capable of efficiently expressing shRNAs with relatively different interfering degrees in avian and mammalian cells, respectively. Our results suggest that ch7SK promoter is an efficient alternative to commercially mouse U6 promoter in shRNA expression with chicken cells, and provide references for furthering functional genome analysis and disease resistant breeding in chicken.
为了将 RNAi 技术应用于鸡，我们开发了一种使用缩短的鸡 7SK（ch7SK）启动子的 RNA 干扰（RNAi）系统。结果表明，克隆的 ch7SK 启动子包含多个 Oct-1 基序、SPH 结构域、PSE 和 TATA 盒，而不包含 CACCC 盒。由 ch7SK 启动子驱动的所有 RNAi 组均表现出显著的平均荧光强度（MFI）降低。在 pch7SK-shEGFP 转染的 DF-EGFP 细胞培养物中，MFI 降低率小于 pmU6-shEGFP。在 pmU6-shEGFP 转染的 Vero-EGFP 细胞培养物中，MFI 显著降低。总之，ch7SK 启动子的必需部分能够在禽和哺乳动物细胞中分别有效地表达具有相对不同干扰程度的 shRNAs。我们的研究结果表明，ch7SK 启动子是在鸡细胞中表达 shRNA 的有效替代商用鼠 U6 启动子的选择，为进一步开展鸡的功能基因组分析和抗病育种提供了参考。
