Abstract
Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, P < 0.05). The ear fibroblasts of the transgenic lambs transfected with the PsiCheck2-VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the “Sleeping Beauty” transposon system is an efficient method to produce transgenic animals.
Highlights
Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals
VP1-short hairpin RNA (shRNA) inhibits the expression of FMDV-VP1
The sense and antisense strands were cloned into the pLL3.7 vector (Fig. 1A), The VP1 gene was obtained by overlapping PCR (Fig. 1B) and cloned into vector psiCheck[2], resulting in a new vector psiCheck2-VP1, which was co-transfected with pLL3.7-NonshRNA or pLL3.7-VP1-shRNA, respectively (4:1), into 293FT
Summary
Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. A conserved VP1-shRNA which targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. They were microinjected into pronuclear embryos to breed transgenic sheep. By targeting VP1 gene-conserved sequences, previous studies have designed one shRNA that can effectively control FMDV infection by inhibiting VP1 gene duplication and further silencing the expression of this protein[6]. In 2015, we successfully bred sixty-one goats, of which seven individuals positively integrated 3D-7414shRNA targeting the 3Dpol gene (which encodes the viral RNA polymerase, a vital element for FMDV replication) in the FMDV genome[14]. Few studies have integrated the SB transposon system with RNAi technology in farm animals, so this study investigated the prospect of producing anti-FMD sheep by utilizing VP1-shRNA coupled with the SB transposon system
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
