Production of prion gene-knockdown cattle by RNA interference(RNAi)technology

利用RNA干扰（RNAi）技术生产朊病毒基因敲除牛

• 批准号: 16310140
• 负责人: OTOI Takeshige
• 金额: $ 10.14万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16310140/
• 关键词: bovine spongiform encephalopathy; prion gene; RNA interference; prion protein; siRNA; cloned animal; クローン動勅

Prion diseases such as bovine spongiform encephalopathy (BSE) in cattle are caused by propagation of misfolded forms (PrP^<se>) of the normal cellular prion protein (PrP^c). By combining RNA interference (RNAi) technology with the somatic cell nuclear transfer (SCNT) method, we attempted to produce transgenic calves with knocked down prion gene (bPRNP). To achieve this, small interfering RNA (siRNA) expression plasmid vectors harboring a Pol III promoter, human U6 (hU6) or tRNA promoter, were constructed. Plasmid DNA was stably introduced into the genome of primary cultured bovine cells. SCNT embryos were produced by inserting the transgenic cell to an enucleated bovine egg, electro-fusing the two, and in vitro culture to the blastocyst stage. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than that of a tRNA group (32%). However, only SCNT embryos with the tRNA promoter could successfully impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP was detected in all six. The transgene of the tRNA vector was detected in two aborted fetuses and two calves. The bPRNP transcript level in the brain from the calf, subjected to euthanasia 20 days after birth, was 38% of that of the control calf as determined by quantitative RT-PCR. The PrP^c level in the brain determined by Western blot, however, was approximately 90% of the control, suggesting that a PrP^c level close to the normal level could be maintained by reduced bPRNP transcripts.

朊病毒疾病如牛海绵状脑病（BSE）是由<se>正常细胞朊病毒蛋白（PrP^c）的错误折叠形式（PrP^c）的繁殖引起的。将RNA干扰（RNAi）技术与体细胞核移植（SCNT）技术相结合，尝试获得朊蛋白基因敲低的转基因犊牛。为了实现这一点，构建了携带Pol III启动子、人U6（hU 6）或tRNA启动子的小干扰RNA（siRNA）表达质粒载体。将质粒DNA稳定地导入原代培养的牛细胞的基因组中。通过将转基因细胞插入去核牛卵中，将两者电融合，并在体外培养至囊胚期来产生SCNT胚胎。在基于hU 6的载体组中，SCNT胚胎发育成囊胚的能力（44-53%）高于tRNA组（32%）。然而，只有带有tRNA启动子的SCNT胚胎可以成功地使受体受精（11次移植中的6次），导致4个流产胎儿，1个死胎和1个活产小牛。在所有6个细胞中均检测到EGFP的表达。在两个流产胎儿和两只小牛中检测到了tRNA载体的转基因。通过定量RT-PCR测定，出生后20天进行安乐死的小牛脑中的bPRNP转录水平为对照小牛的38%。然而，通过蛋白质印迹法测定的大脑中PrP^c水平约为对照组的90%，这表明通过减少bPRNP转录物可以维持接近正常水平的PrP^c水平。