Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference
Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference
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通过RNA干扰开发对BVDV-1不同亚基因型具有增强抗性的绵羊肾细胞
DOI：
10.1016/j.jviromet.2015.03.014
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发表时间：
2015-06-15
影响因子：
3.1
通讯作者：
Chen, Chuangfu
中科院分区：
文献类型：
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作者：
Ni, Wei;Qiao, Jun;Chen, Chuangfu
牛病毒性腹泻病毒(BVDV)应该是牛羊养殖业普遍存在的病毒病原。这种病原体造成了严重的经济损失。我们先前已经证明，质粒介导的双短发夹状RNA(ShRNA)能有效地抑制牛肾上皮(MDBK)细胞中BVDV的复制。在本研究中，我们将双shRNA系统导入绵羊成纤维细胞，并获得了转基因细胞克隆。这些转基因成纤维细胞进一步用于体细胞核移植。三只羔羊在足月出生，但出生后不久就死亡了。用聚合酶链式反应证实shRNA已整合到克隆羊基因组中，用实时荧光定量聚合酶链式反应检测到转基因绵羊中有shRNA的表达。从转基因绵羊中分离出肾上皮细胞，用BVDV-1a、BVDV-1b和BVDV-1c多种亚型进行攻击。在转基因肾上皮细胞中表达的双shRNA以交叉耐药的方式显著抑制BVDV复制。我们的结果表明，转基因RNAi可能是一种有用的工具，用于制备对BVDV抗性增强的转基因动物。(C)2015爱思唯尔B.V.保留所有权利。
Bovine viral diarrhea virus (BVDV) should be a ubiquitous viral pathogen to the cattle and sheep industry. This pathogen is responsible for severe economic losses. We previously showed that plasmid-mediated dual short hairpin RNA (shRNA) efficiently inhibit BVDV replication in bovine kidney epithelial (MDBK) cells. In this study, we delivered the dual shRNA system to sheep fibroblasts and generated transgenic cell colonies. These transgenic fibroblasts were further used for somatic cell nuclear transfer (SCNT). Three lambs were born at full term, but perished soon after birth. Integration of shRNA into the genome of cloned sheep was confirmed by PCR and expression of shRNA in transgenic sheep was confirmed by real-time PCR. Kidney epithelial cells were isolated from transgenic sheep and challenged with multiple BVDV subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). The dual shRNA expressed in transgenic kidney epithelial cells significantly inhibited BVDV replication in a cross-resistance manner. Our results showed that transgenic RNAi might be a useful tool for preparation of transgenic animals with increased resistance to BVDV. (C) 2015 Elsevier B.V. All rights reserved.