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

疯牛病，如牛海绵状脑病(BSE)，是由正常细胞内蛋白(PrP^c)的错误折叠形式(PrP^和lt；Se和gt；)的繁殖引起的。本研究将RNA干扰(RNAi)技术与体细胞核移植(SCNT)技术相结合，试图获得具有敲除蛋白基因(BPRNP)基因的转基因小牛。为此，构建了含有Pol III启动子、人U6(HU6)启动子或tRNA启动子的小干扰RNA(SiRNA)表达载体。将质粒DNA稳定地导入原代培养的牛细胞基因组中。将转基因细胞插入去核的牛卵中，将两者电融合，并体外培养至囊胚期，即可获得SCNT胚胎。在HU6载体组中，SCNT胚胎发育到囊胚的能力(44-53%)高于tRNA组(32%)。然而，只有带有tRNA启动子的SCNT胚胎才能成功使受体受孕(11次移植中有6次)，导致4例流产、1例死产和1例活产小牛。六种细胞均检测到绿色荧光蛋白的表达。在两个流产的胎儿和两个牛犊中检测到了tRNA载体的转基因。用定量RT-PCR方法检测出生后20天安乐死后小牛脑组织中bPRNP的转录水平是对照组的38%。然而，通过Western印迹检测，大脑中PrP^c的水平大约是对照的90%，这表明bPRNP转录本的减少可以维持接近正常水平的PrP^c水平。

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.