Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

Abstract

BackgroundAlthough 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.Principal FindingHere 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 TCID50 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.ConclusionRNAi-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.

Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

“…Lentivirus-based shRNA vectors were constructed as described previously (21). The sequences of these shRNA oligonucleotides are listed as follows: PIAS3-1, 59-GGAGCCAAATGTGATTATA-39; PIAS3-2, 59-CATCCAA-GGTTTAGATTTA-39; PIAS3-3, 59-CAAGAAGGCTCCCTATGAA-39; Scramble (control shRNA), 59-GACGATGATTCGTATGTAA-39.…”

Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA

“…Tracheal tissue was obtained from a fetal Holstein cow of the age of 4 months gestation under aseptic conditions (Wang et al, 2012). Th e bovine fetal primary tracheal epithelial cells were separated, cultured, and cryopreserved according to the methods described by Hauser et al (2013).…”

“…Lentivirus-based shRNA vectors were constructed as described previously (Wang et al, 2012). Th e sequences of these of shRNA oligonucleotides are RNAi-Mx1-A1: 5'-GCAACCTGTACAGCCAATATGTTCAAGAGACATAT TGGCTGTACAGGTTGCTTTTTTGT-3' and RNAi-Mx1-A2: 5 ' -C TAG AC A A A A A AG C A AC C T G TAC AG C C A ATA TGTCTCTTGAACATATTGGCTGTACAGGTTGC-3'; RNAiMx1-B1: 5'-GGAATGAAGACGAGTGGAAAGTTCAAG AGACTTTCCACTCGTCTTCATTCCTTTTTTGT-3' and RNAi-Mx1-B2: 5'-CTAGACAAAAAAGGAATGAAGA CGAGTGGATCTCTTGAACTTTCCACTCGTCTTCATTCC-3'; RNAi-Mx1-C1: 5'-GGATCAGTCATGAGCTGATTATCAAG AGATAATCAGCTCATGACTGATCCTTTTTTGT-3' and RNAiMx1-C2: 5'-CTAGACAAAAAAGGATCAGTCATGAGCTGA T TATCTCT TGAATAATCAGCTCATGACTGATCC-3'.…”

“…PCR primer pairs were used as follows: Mx1-P1:5'-ATAAGGTTCATTCTGACTTG-3' and Mx1-P2: 5'-GCCGCACCTTCTCTTCAT-3' for bovine Mx1 RNA, β-actin-P1: 5'-AGCAAGCAGGAGTACGATGAG-3' and β-actin-P2: 5'-TGCCAATCTCATCTCGTTTTC-3' for β-actin. As a control, 10-fold dilutions of 10 7 copies of pcDNA3.1-Flag-Mx1 vector and T-actin vector (Wang et al, 2012), respectively, were used. All further computations were done using ABI Prism 7000 SDS Soft ware (Applied Biosystems).…”

Construction and Detection of Recombinant Adeno-Associated Virus Co- Expressing PRRSV GP5, M Proteins and shRNA

“…The administration of virus-sequence-targeted short interfering RNA (siRNA) with a length of 21-33 nucleotides has inhibited viral replication effects in mammalian cells and animals [4]. Short hairpin RNA (shRNA) can also downregulate the expression of viral genes and protect animals from virus challenge through binding to virus-specific mRNA [5]. RNAi has already been employed as a mechanism of therapeutic gene silencing and anti-viral therapy.…”