Genetically modified pigs are protected from classical swine fever virus

Fig 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)…

Fig 2. Generation of F0-generation TG pigs expressing the targeting siRNA. (A) Genomic PCR analysis of the F0-generation to identify TG pigs. P1, P2, P4, P5, P6, P8, P11 and P12 indicate TG pigs, and P3, P7, P9 and P10 indicate NTG pigs. M: D2000. (B) Schematic for Southern blotting. The hybridization signals indicated that the shRNA gene was successfully integrated into the pRosa26 locus in the TG pigs. M: DNA maker. PC: positive control plasmid. P3 and P7 indicate NTG pigs, and P1 and P2 indicate TG pigs. (C) The relative expression levels of the targeting siRNA in various tissues and organs from TG pigs were determined by qPCR. The expression levels were analysed using the unpaired t-test (*p<0.05; **p<0.01). Error bars represent the SEM, n = 3. TG: transgenic pigs. NTG: wild-type pigs. (D) Virus resistance in three types of isolated primary cells from TG and NTG pigs was examined by IFA. The cells cultured in 24-well plates were inoculated with 200 TCID50 of CSFV (Shimen strain). At 72 hours post infection, the CSFV-infected cells were incubated with an E2-specific antibody (PAb) and then stained with fluorescein isothiocyanate (FITC)-labelled goat anti-pig IgG (1:100). Cells were…

Fig 3. Off-target site analysis. (A) The target site (T) and ten predicted off-target sites (OT) for sgRNA91/Cas9. OT1~OT10 represents 10 potential off-target sites, and T represents target site. (B) T7E1 cleavage assays for the target site and the ten potential off-target sites; M, DL2000; (C) Sanger sequencing analyses of PCR amplicons that spanning the target sites. The cleavage sites are labeled with red arrow. (D) Sanger sequencing analyses of PCR amplicons that spanning the potential off-target sites. The potential cleavage sites are labeled with red arrow.

Fig 4. Generation of F1-generation TG pigs. (A) Photographs show one of the F1-generation TG piglets. (B) The knock-in events in newborn TG piglets were confirmed by genomic PCR. Pigs 0042, 0047, 0049, 0050, 0058 and 0059 are TG pigs, and pigs 0041, 0044, 0045, 0060 and 0061 were NTG. M, DNA marker (D2000, Tiangen, Beijing, China). (C) The relative expression levels of the targeting siRNA in F0- and F1-transgenic cells were compared by qPCR. Targeting siRNA expression levels were analysed with the unpaired t-test (ns. not significant). Error bars represent the SEMs, n = 3. TG-F1 represents F0-generation TG pigs. TG-F1 represents F1-generation TG pigs. (D) qPCR results show the relative expression of viral RNA in NTG cells, F0-generation TG cells and F1-generation TG cells. Viral RNA expression was analysed with an unpaired t-test (ns. not significant; ***p<0.001). Error bars represent the SEMs, n = 3.

Fig 5. Experimental grouping and relative clinical monitoring data of the challenged pigs. (A) Schematic depiction of the in-contact challenge assay. The black box indicates a separate room. Different colours indicate different types of pigs. Each room included one non-TG pig that was injected with CSFV (NTG-In), three NTG pigs and three TG pigs. (B) Mortality and viremia in challenged pigs. The survival time of each pig is indicated by the length of the green bar. Red squares indicate the collection of blood samples. The terminal block colour indicates day and cause of death (black, found dead; blue, pigs (TG-4 and TG-5) euthanized for comparison to the dead NTG pig). The numbers for each day are the lg values of viral RNA copies present in the blood samples.

Fig 6. TG pigs exhibit antiviral responses during CSFV infection. (A) Survival curves of the challenged pigs during in-contact infection. Different curves indicate different pigs. The blue curve indicates the NTG pigs and the red curve indicates the TG pigs. The pigs in the TG group survived significantly longer than those in the NTG group. (B) The rectal temperatures of the pigs following challenge with CSFV (TG group, n = 6; NTG group, n = 6). The rectal temperatures were measured daily until the animals died. Error bars represent the standard deviations of at least 3 biological replicates. (C) Body weight curves for the challenged pigs during in-contact infection. Error bars represent the standard deviations of at least 3 biological replicates. (D) Viral replication curves in the challenged pigs. CSFV genome copies in the blood samples are presented according to the standard curve. Data in the panels are presented as the mean ± SE. Error bars represent the standard deviations of at least 3 biological replicates. (E) Serum was collected from pigs on 0, 4, 8 and 16 dpi to analyse antibodies against CSFV using a commercial CSFV Antibody Test Kit (IDEXX). ELISA plates were read using a…