RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells

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.

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

“…This highlights the potential for the combination of the Sleeping Beauty transposon with RNAi to generate knockdown donor cells for animal cloning. This combined system was also used to generate transgenic sheep with resistance to FMDV by knocking down the FMDV- VP1 gene (Deng et al, 2017). After pronuclear microinjection, eight out of 92 generated lambs showed positive integration of VP1 -shRNA.…”

“…Tongue epithelium cells isolated from transgenic goats carrying shRNA against the FMDV 3Dpol gene showed effective resistance after FMDV challenge (Li et al, 2015). Moreover, ear fibroblasts isolated from transgenic lambs that carried shRNA against the FMDV- VP1 gene showed a significant inhibitory effect on the VP1 gene (Deng et al, 2017). These studies highlight the possibilities for the use of RNAi strategies to confer potential disease resistance in farm animals.…”

Silencing of the foot-and-mouth disease virus internal ribosomal entry site by targeting relatively conserved region among serotypes

“…These features make this construct promising in terms of solving several current challenges with FMDV vaccination. Moreover, this lenti-shRNA expression vector could be applicable to transduction in embryos for construction of transgenic animals [21]. For establishment of therapeutic vectors in vivo in future studies, it will be necessary to use vectors other than lentiviruses, e.g., adenovirus vectors [22, 23] and adeno-associated virus vectors [24].…”

Genome Engineering in Livestock: Recent Advances and Regulatory Framework

“…The transposase binds to the ITRs region forming a synaptic complex that leads to the transposon being introduced into a genomic consensus site (Ivics et al, 2009;Mun˜oz-Lo´pez and Garcı´a-Pe´rez, 2010). Sleeping Beauty and piggyBac transposon systems were used to produce transgenic livestock such as cattle (Garrels et al, 2016;Yum et al, 2016;Yum et al, 2018), sheep (Deng et al, 2017), goats (Bai et al, 2017) and pigs (Garrels et al, 2011;Carlson et al, 2011;Ivics et al, 2014). The transposon system is considered as valuable tool for the efficient genomic integration and stable expression of transgenes with germline transmission capability.…”