Ni Wei, Qiao Jun, Ma Qiman, Wang Jiangde, Wang Dawei, Zhao Xinxia, Cao Yang, Li Qifeng, Hu Shengwei, Chen Chuangfu
College of Life Sciences, Shihezi University, Shihezi, Xinjiang 832003, China.
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China.
J Virol Methods. 2015 Jun 15;218:66-70. doi: 10.1016/j.jviromet.2015.03.014. Epub 2015 Mar 23.
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.
牛病毒性腹泻病毒（BVDV）对于牛羊产业来说应该是一种普遍存在的病毒病原体。这种病原体造成了严重的经济损失。我们之前表明，质粒介导的双短发夹RNA（shRNA）能有效抑制牛病毒性腹泻病毒在牛肾上皮（MDBK）细胞中的复制。在本研究中，我们将双shRNA系统导入绵羊成纤维细胞并产生了转基因细胞集落。这些转基因成纤维细胞进一步用于体细胞核移植（SCNT）。三只羔羊足月出生，但出生后不久就死亡了。通过PCR确认了shRNA整合到克隆绵羊的基因组中，并通过实时PCR确认了shRNA在转基因绵羊中的表达。从转基因绵羊中分离出肾上皮细胞，并用多种BVDV亚型（BVDV-1a、BVDV-1b和BVDV-1c）进行攻击。转基因肾上皮细胞中表达的双shRNA以交叉抗性方式显著抑制了BVDV的复制。我们的结果表明，转基因RNA干扰可能是制备对BVDV具有增强抗性的转基因动物的有用工具。
