RNA polymerase III is an essential enzyme in eukaryotes for synthesis of tRNA, 5S rRNA, and other small nuclear and cytoplasmic RNAs. Thus, RNA polymerase III promoters are often used in small hairpin RNA (shRNA) expression. In this study, the porcine H1, U6, and 7SK RNA polymerase III type promoters were cloned into a pcDNA3.1( +) expression vector containing a shRNA sequence targeting enhanced green fluorescent protein (EGFP). PK and DF-1 cells were cotransfected with the construction of recombinant interference expression vector and the EGFP expression vector, pEGFP-N1. The average fluorescence intensity of EGFP in transfected cells was measured by fluorescence microscopy and flow cytometry. Real-time PCR was used to detect expressed shRNAs and the relative expression of EGFP, to confirm the activity of the promoters. The results showed that the activity of porcine 7SK promoter is stronger than the U6 promoter, which is in turn stronger than porcine H1. While the high levels of expression of the U6 and 7SK promoters saturate the shRNAs level in the host cell, which can cause cytotoxicity and tissue damage. Therefore, porcine H1 promoter is effective for expression of shRNA, and may be an excellent tool to knockdown gene expression in pigs for functional genomics studies. The results also lay a foundation for the development of porcine RNAi technology and genetically modified porcine research.

中文翻译：

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鉴定和比较猪H1，U6和7SK RNA聚合酶III启动子的短发夹RNA表达。

RNA聚合酶III是真核生物中合成tRNA，5S rRNA以及其他小核和细胞质RNA所必需的酶。因此，RNA聚合酶III启动子经常用于小发夹RNA（shRNA）表达。在这项研究中，将猪H1，U6和7SK RNA聚合酶III型启动子克隆到一个pcDNA3.1（+）表达载体中，该载体包含靶向增强绿色荧光蛋白（EGFP）的shRNA序列。用重组干扰表达载体和EGFP表达载体pEGFP-N1的构建共转染PK和DF-1细胞。通过荧光显微镜和流式细胞术测量转染细胞中EGFP的平均荧光强度。使用实时荧光定量PCR检测表达的shRNA和EGFP的相对表达，以确认启动子的活性。结果表明，猪7SK启动子的活性强于U6启动子，而U6启动子又强于猪H1。U6和7SK启动子的高水平表达会饱和宿主细胞中的shRNAs水平，这可能导致细胞毒性和组织损伤。因此，猪H1启动子对于shRNA的表达有效，并且可能是敲低猪中基因表达以进行功能基因组学研究的极佳工具。该结果也为猪RNAi技术的发展和转基因猪研究奠定了基础。因此，猪H1启动子对于shRNA的表达有效，并且可能是敲低猪中基因表达以进行功能基因组学研究的极佳工具。该结果也为猪RNAi技术的发展和转基因猪研究奠定了基础。因此，猪H1启动子对于shRNA的表达有效，并且可能是敲低猪中基因表达以进行功能基因组学研究的极佳工具。该结果也为猪RNAi技术的发展和转基因猪研究奠定了基础。