State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China.
Mol Biol Rep. 2010 Oct;37(7):3193-8. doi: 10.1007/s11033-009-9900-0. Epub 2009 Oct 11.
PRNP is the gene encoding prion protein whose misfolded and β-sheet-rich isoform is the infectious agent of transmissible spongiform encephalopathy (TSE). TSE, also called prion diseases, cause fatal neurodegenerative and transmissible disorders in human and animals. Among these diseases, bovine spongiform encephalopathy (BSE) has tremendous impact on economy and human health in the world. In the present study, we hypothesize suppression of the PRNP gene expression could raise resistance to BSE in cattle by using vector-based small interfering RNA (siRNA) expression systems. Therefore, the objective was to screen effective DNA-encoding short hairpin RNAs (shRNAs) which could knockdown the PRNP gene expression in bovine fibroblast cells. Human U6 promoter was employed to drive shRNA transcription from the DNA vector, and seven shRNAs, that designed to target coding region and 3' untranslated region of the PRNP gene, were selected. Four out of seven shRNAs tested were found to be effective in inhibiting the PRNP gene expression, and the most significant suppression level was as much as 62.9% evidenced by real-time RT-PCR. Furthermore, the protein abundance was obviously reduced compared to the control. Overall, the present study demonstrated that vector-based siRNA expression systems is an efficient approach to knockdown the PRNP gene expression in bovine fibroblast cells and thereby provide donor cells for somatic cell nuclear cloning to produce cattle that is resistant to prion related diseases.
PRNP 是编码朊病毒蛋白的基因，其错误折叠和富含β-折叠的异构体是传染性海绵状脑病（TSE）的感染剂。TSE 也称为朊病毒病，导致人和动物致命的神经退行性和传染性疾病。在这些疾病中，牛海绵状脑病（BSE）对世界经济和人类健康产生了巨大影响。在本研究中，我们假设通过基于载体的小干扰 RNA（siRNA）表达系统抑制 PRNP 基因表达可以提高牛对 BSE 的抗性。因此，本研究的目的是筛选能够在牛成纤维细胞中敲低 PRNP 基因表达的有效 DNA 编码短发夹 RNA（shRNA）。用人 U6 启动子驱动 shRNA 从 DNA 载体转录，选择了 7 条针对 PRNP 基因编码区和 3'非翻译区的 shRNA。测试的 7 条 shRNA 中有 4 条被发现能有效抑制 PRNP 基因表达，实时 RT-PCR 结果显示抑制水平最高可达 62.9%。此外，与对照组相比，蛋白质丰度明显降低。总之，本研究表明，基于载体的 siRNA 表达系统是一种有效抑制牛成纤维细胞中 PRNP 基因表达的方法，并为生产对朊病毒相关疾病具有抗性的牛提供了体细胞核移植的供体细胞。
