Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.
BACKGROUND
Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.


RESULTS
Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50) of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.


CONCLUSIONS
RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.
Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan, People's Republic of China.
Hongmei
H
Wang
Jianming
J
Wu
Xiao
X
Liu
Hongbin
H
He
Fangrong
F
Ding
Hongjun
H
Yang
Lei
L
Cheng
Wenhao
W
Liu
Jifeng
J
Zhong
Yunping
Y
Dai
Guangpeng
G
Li
Chengqiang
C
He
Li
L
Yu
Jianbin
J
Li
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D006224:Cricetinae; D004847:Epithelial Cells / Q000166:cytology*; D005536:Foot-and-Mouth Disease / Q000517:prevention & control / Q000821:virology*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008297:Male; D009865:Oocytes / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014059:Tongue / Q000378:metabolism; D014779:Virus Replication
D034741:RNA, Small Interfering