No abstract

Classical 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.

Background: Apoptosis is recognized as an important mechanism in contrast-induced nephropathy (CIN). As tetramethylpyrazine (TMP) has been recently found to be renoprotective and anti-apoptotic in multiple kidney injuries, we hypothesized that TMP would prevent CIN. Methods: An experimental model of CIN was established in rats. Serum creatinine, blood urea nitrogen, plasma cystatin C, urinary N-acetyl-β-glucosaminidase, and urinary γ-glutamyl transpeptidase were measured to evaluate kidney function. Apoptosis was assessed by transmission electron microscopy, transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and poly-ADP-ribose polymerase cleavage. Fork-head box O1 transcriptional factor (FoxO1) mRNA expression was evaluated by quantitative real-time PCR. Phospho-p38 mitogen-activated protein kinase (MAPK) protein expression was assessed by immunohistochemistry and Western blotting. Results: TMP significantly attenuated the resulting renal dysfunction and renal tubular cell apo-ptosis. Mechanistically, TMP decreased the expression of phospho-p38 MAPK protein and attenuated the increased FoxO1 mRNA and nuclear protein expression. In addition, TMP inhibited inducible nitric oxide synthase and Bax protein expression while it upregulated Bcl-2. Conclusion: In summary, this study demonstrated the protective role of TMP against CIN and indicated the effects of TMP may be mediated by the inhibition of p38 MAPK and FoxO1 pathways. Thus, TMP may be a new potential therapeutic agent to prevent CIN.

CRISPR/Cas9 has emerged as one of the most popular genome editing tools due to its simple design and high efficiency in multiple species. Myostatin (MSTN) negatively regulates skeletal muscle growth and mutations in myostatin cause double-muscled phenotype in various animals. Here, we generated myostatin mutation in Erhualian pigs using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. The protein level of myostatin precursor decreased dramatically in mutant cloned piglets. Unlike myostatin knockout Landrace, which often encountered health issues and died shortly after birth, Erhualian pigs harboring homozygous mutations were viable. Moreover, myostatin knockout Erhualian pigs exhibited partial double-muscled phenotype such as prominent muscular protrusion, wider back and hip compared with wild-type piglets. Genome editing in Chinese indigenous pig breeds thus holds great promise not only for improving growth performance, but also for protecting endangered genetic resources.

Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.