Abstract
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.
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Acknowledgements
We thank Hong-Yan Chen (Chinese Academy of Agriculture Sciences Harbin Veterinary Research Institute) for providing guide.
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This work was supported by the grant from the State Key Laboratory of Veterinary Biotechnology Program [SKLVBP201801]. Qing-Wen Meng are associated with the funding sources.
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HCY and QWM designed the experiments. HCY and WW performed the experiments. HCY wrote the paper. XYC analyzed the data and drew the graphs.
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Yin, HC., Chen, XY., Wang, W. et al. Identification and comparison of the porcine H1, U6, and 7SK RNA polymerase III promoters for short hairpin RNA expression. Mamm Genome 31, 110–116 (2020). https://doi.org/10.1007/s00335-020-09838-0
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DOI: https://doi.org/10.1007/s00335-020-09838-0