State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, PR China.
J Biotechnol. 2013 Feb 20;163(4):377-85. doi: 10.1016/j.jbiotec.2012.10.018. Epub 2012 Nov 29.
The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT). However, the enrichment efficiency of the gene targeting vector was low. Here, we constructed a novel promoterless gene targeting vector to sequentially disrupt the PRNP gene in bovine fibroblast cells and generate gene targeted cattle by SCNT. The enrichment efficiency of the novel vector was 100% and 60%, respectively. After nuclear transfer, no significant difference was found in the rate of cleavage and blastocyst formation between the knockout and wild type cloned embryos. One PRNP⁺/⁻ calf was born with no obvious abnormal development by now. Fusion RT-PCR and real-time PCR showed one allele of the PRNP gene was functionally disrupted, and the mRNA expression reduced dramatically in the PRNP⁺/⁻ cattle. The reconstituted PRNP⁻/⁻ embryos showed double alleles disruption, and no difference in the rate of cleavage and blastocyst formation.
PRNP 基因编码一种名为朊病毒的细胞蛋白，其错误折叠形式与哺乳动物的多种神经病变疾病有关，如牛海绵状脑病（BSE）。BSE 给世界经济和人类健康带来了毁灭性的影响。最近，有几个研究小组采用基因打靶策略，通过体细胞核移植（SCNT）破坏牛成纤维细胞中的 PRNP 基因，从而产生抗 BSE 的牛。然而，基因打靶载体的富集效率较低。在这里，我们构建了一种新型无启动子基因打靶载体，以顺序破坏牛成纤维细胞中的 PRNP 基因，并通过 SCNT 产生基因靶向牛。新型载体的富集效率分别为 100%和 60%。核移植后，敲除和野生型克隆胚胎的卵裂率和囊胚形成率没有明显差异。到目前为止，已经有一只 PRNP⁺/⁻小牛出生，没有明显的异常发育。融合 RT-PCR 和实时 PCR 显示 PRNP 基因的一个等位基因失活，PRNP⁺/⁻牛的 mRNA 表达显著降低。重构的 PRNP⁻/⁻胚胎显示出两个等位基因缺失，卵裂率和囊胚形成率没有差异。
