JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2014 Vol.15 No.5 P.466-473

Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways* #

Author(s): Zhen Dai¹, Rong Wu¹, Yi-cheng Zhao^1,2, Kan-kan Wang¹, Yong-ye Huang¹, Xin Yang¹, Zi-cong Xie¹, Chang-chun Tu³, Hong-sheng Ouyang¹, Tie-dong Wang¹, Da-xin Pang¹
Affiliation(s): 1. Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, Changchun 130062, China; more
Corresponding email(s): tdongw@sohu.com
Key Words: MicroRNA pathway, shRNA-transgenic pigs, Classical swine fever virus (CSFV), Blastocyst formation, Early lethality

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Zhen Dai, Rong Wu, Yi-cheng Zhao, Kan-kan Wang, Yong-ye Huang, Xin Yang, Zi-cong Xie, Chang-chun Tu, Hong-sheng Ouyang, Tie-dong Wang, Da-xin Pang. Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways[J]. Journal of Zhejiang University Science B, 2014, 15(5): 466-473.

@article{title="Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways",
author="Zhen Dai, Rong Wu, Yi-cheng Zhao, Kan-kan Wang, Yong-ye Huang, Xin Yang, Zi-cong Xie, Chang-chun Tu, Hong-sheng Ouyang, Tie-dong Wang, Da-xin Pang",
journal="Journal of Zhejiang University Science B",
volume="15",
number="5",
pages="466-473",
year="2014",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1400001"
}

%0 Journal Article
%T Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways
%A Zhen Dai
%A Rong Wu
%A Yi-cheng Zhao
%A Kan-kan Wang
%A Yong-ye Huang
%A Xin Yang
%A Zi-cong Xie
%A Chang-chun Tu
%A Hong-sheng Ouyang
%A Tie-dong Wang
%A Da-xin Pang
%J Journal of Zhejiang University SCIENCE B
%V 15
%N 5
%P 466-473
%@ 1673-1581
%D 2014
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1400001

TY - JOUR
T1 - Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways
A1 - Zhen Dai
A1 - Rong Wu
A1 - Yi-cheng Zhao
A1 - Kan-kan Wang
A1 - Yong-ye Huang
A1 - Xin Yang
A1 - Zi-cong Xie
A1 - Chang-chun Tu
A1 - Hong-sheng Ouyang
A1 - Tie-dong Wang
A1 - Da-xin Pang
J0 - Journal of Zhejiang University Science B
VL - 15
IS - 5
SP - 466
EP - 473
%@ 1673-1581
Y1 - 2014
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1400001

Abstract
Chinese Summary
Academic Network
Reviewer Comment

Abstract: RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.

shRNA转基因猪中miRNA通路饱和引起的早期致死性

研究目的：通过制备抗猪瘟病毒shRNA转基因猪，探索shRNA对猪胎儿成纤维细胞、克隆猪早期胚胎发育以及转基因猪的影响。
创新要点：基于稳定表达shRNA的猪胎儿成纤维克隆细胞及shRNA转基因克隆猪制备的结果，发现shRNA对克隆猪早期囊胚发育阶段无明显毒性，并首次报道了在shRNA转基因动物中，shRNA引起体内miRNA通路过饱和及动物致死性等毒性。
研究方法：实验构建了多种抗猪瘟病毒shRNA表达载体，转染猪胎儿成纤维细胞并筛选得到细胞克隆，结合体细胞核移植技术制备了shRNA转基因猪。使用实时荧光定量聚合酶链式反应（PCR）和蛋白质印迹法（Western blotting）检测了细胞及个体水平siRNA的表达量、干扰素反应相关基因、内源miRNA及通路因子的表达量，同时检测了shRNA克隆细胞囊胚率。
重要结论：shRNA能有效抑制猪瘟病毒的复制。在猪胎儿成纤维细胞水平上，稳定表达shRNA能引起细胞内干扰素反应、miRNA通路过饱和等副作用；在克隆猪发育阶段，shRNA对克隆猪早期囊胚发育阶段无明显毒性；在shRNA转基因克隆猪个体水平上，shRNA引起克隆猪体内miRNA通路过饱和及克隆猪早期易致死等毒性。

关键词：致死性；shRNA转基因克隆猪；microRNA通路；猪瘟病毒；囊胚发育

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shRNA转基因猪中miRNA通路饱和引起的早期致死性

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