Genetically modified pigs are protected from classical swine fever virus
Tuesday, 2018/12/25 | 08:01:29
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Zicong Xie, Daxin Pang, Hongming Yuan, Huping Jiao, Chao Lu, Kankan Wang, Qiangbing Yang, Mengjing Li, Xue Chen, Tingting Yu, Xinrong Chen, Zhen Dai, Yani Peng, Xiaochun Tang, Zhanjun Li, Tiedong Wang, Huancheng Guo, Li Li, Changchun Tu, Liangxue Lai, Hongsheng Ouyang PLOS Pathogens; Published: December 13, 2018 https://doi.org/10.1371/journal.ppat.1007193 AbstractClassical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research. Author summaryClassical swine fever (CSF), caused by classical swine fever virus (CSFV), and is a highly contagious, often fatal porcine disease that causes significant economic losses. Due to the economic importance of this virus to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread, and it is only a matter of time before the virus is reintroduced and the next round of disease outbreaks occurs. These findings highlight the necessity and urgency for developing effective approaches to eradicate the challenging CSFV. In this study, we successfully produced anti-CSFV TG pigs by combining RNAi technology and CRISPR/Cas9 technologies, and viral challenge results confirmed that these TG pigs could effectively limit the replication of CSFV in vivo and in vitro. Additionally, we confirmed that the disease resistance traits in the TG founders were stably transmitted to their F1-generation offspring. Altogether, our work reported the combinational application of CRISPR/Cas9 and RNA interference (RNAi) technology in the generation of anti-CSFV TG pigs, it provided an alternative strategy to change the virus. The results of this study suggested that these TG pigs offered potential benefits over commercial vaccination and reduced CSFV-related economic losses.
See https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007193
Figure 1: Selection of antiviral cell clones. (A) Reduction of viral genome replication in siRNA-transfected cells was further assessed by real time PCR at 72 h post-infection. Error bars represent the SEMs, n = 3. (B) Genomic PCR analysis confirmed the knock-in events at the pRosa26 locus by using specific primers (Table 4). The KI-1 primers were used to determine homozygosity or heterozygosity, the KI-2 primers amplified the 5’ junction, and the KI-3 primers amplified the 3’ junction junction. The blue arrows indicate target amplicons, and the corresponding sizes of the PCR amplicons are 990, 932 and 1291 bp. Lanes 2–10 represent the positive shRNA knock-in PFF clones. NC: negative control (wild-type PFFs). M: D2000. The corresponding sequences of these primers are listed in Table 4. (C) Inhibition of viable viral production in shRNA knock-in cell clones (PFF and PK-15 cell clones) was further assessed by real-time PCR at 72 h post-infection. The copies of the viral genomes were analysed using the unpaired t-test. Error bars represent the SEMs, n = 3. Sh-C3: sh-C3 knock-in cell clone. Sh-C6: sh-C6 knock-in cell clone. Sh-Scr: scrambled shRNA knock-in cell clone. WT: wild-type PFFs. (D) Expression of the two targeting siRNAs in the corresponding transgenic PFF clones was confirmed by RT-PCR. M: 50bp DNA ladder. C1: wild-type PFFs. C2: scrambled shRNA transgenic PFF clones. #44: shRNA-C3 transgenic PFF clones. #65: shRNA-C6 transgenic PFF clones. The size of the target amplicons was 76 bp. Endogenous U6 was used as an RNA quality and loading control. |
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