Viral infection resistance conferred on mice by siRNA transgenesis

Abstract

RNA interference is an attractive strategy to fight against viral diseases by targeting the mRNA of viral genes. Most studies have reported the transient delivery of small interfering RNA or small hairpin (shRNA) expression constructs. Here, we present the production of transgenic mice stably expressing shRNA or miRNA targeting the IE180 mRNA (immediate early gene) of the pseudorabies virus (PRV) which infects mice and farm animals. We firstly designed non-retroviral shRNA or miRNA expression vectors. Secondly, we selected the most efficient shRNA construct that targeted either the 5'part or 3'UTR of the IE mRNA and was able to knockdown the target gene expression in cultured cells, by measuring systematically the shRNA content and comparing this with the interfering effects. We then produced four lines of transgenic mice expressing different amounts of shRNA or miRNA in the brain but without signs of stimulation of innate immunity. Lastly, we tested their resistance to PRV infection. In all transgenic lines, we observed a significant resistance to viral challenge, the best being achieved with the shRNA construct targeting the 3'UTR of the IE gene. Viral DNA levels in the brains of infected mice were always lower in transgenic mice, even in animals that did not survive. Finally, this work reports an effective strategy to generate transgenic animals producing shRNA from non-retroviral expression vectors. Moreover, these mice are the first transgenic animal models producing shRNA with a significant antiviral effect but without any apparent shRNA toxicity.

Induction of Body Weight Loss through RNAi-Knockdown of APOBEC1 Gene Expression in Transgenic Rabbits

“…A construct encompassing a shRNA expressing gene (rbapobec1-shRNA, Figure 1) was therefore introduced by microinjection in the pronuclei of fertilized unicellular rabbit embryos. The sequence of the shRNA targeting the rabbit APOBEC1 mRNA was chosen among a set of sequences designed by using the OligoWalk tool [11] after assessment of its high efficiency by using an in vitro test as previously described [12] (Figure S1). …”

“…Here, we produced transgenic rabbits expressing permanently a small interfering RNA (siRNA) targeting the rabbit intestinal APOBEC1 gene. This was achieved through the introduction in the rabbit genome of a DNA construct expressing a small hairpin RNA by using a strategy that we had followed in a previous study [12]. This strategy had the advantage to provoke the sustained production of the siRNA, and a moderate but significant and permanent decrease of the rabbit APOBEC1 gene expression.…”

“…The shRNA transcription unit was constructed from synthetic DNA fragments (Eurofins, Ebersberg, Germany). This H1-rbapobec1-shRNA gene was then inserted into the pM10 vector [12] at the enzymatic BsaBI-ClaI restriction sites as presented in Figure 1. The final construct used for microinjection encompassed the DNA fragment included between the two NotI restriction sites.…”

Impacts of genetically modified animals on the ecosystem and human activities

“…Using different tools it was possible, in other projects, to reduce cat fish sensitivity to bacteria (Dunham, 2009); to generate mice resistant to the pig Aujeszki disease (Daniel-Carlier et al, 2012;Ono et al, 2004); to establish lines of chicken resistant to the influenza virus H5N1 (Lyall et al, 2011); and to obtain cows potentially resistant to mad cow disease (Denning et al, 2001). Three proteins: human lactoferrin (hLf), human lysozyme (hLys), and lysostaphin are known to have antibacterial activities.…”

Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference

“…These RNAi strategies have been used successfully for suppressing the replication of human and animal viruses, including hepatitis B virus (HBV) (Kayhan et al, 2007), human immunodeficiency virus type (HIV) (Coburn and Cullen, 2002), porcine reproductive and respiratory syndrome virus (PRRSV) (Luo et al, 2013) and foot-and-mouth disease virus (FMDV) (De los Santos et al, 2005;Lv et al, 2009;Xu et al, 2012). Furthermore, we and others reported that transgenic animals expressing shRNA against virus genes showed a significant resistance to viral challenge (Pengyan et al, 2010;Li et al, 2014;Daniel-Carlier et al, 2013;Du et al, 2014). More intriguingly, one study demonstrated that transgenic shRNA chickens when challenged with H5N1 highly pathogenic avian influenza virus (A/turkey/Turkey/1/2005) did not result in transmission.…”