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

Figures

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  Figure 1. Inhibitory effects of VP1-shRNA on FMDV-VP1. (A) Schematic map of the pLL3.7-VP1-shRNA expression vector. (B) The VP1 gene amplification using overlapping PCR; 1: a-h, 2: a-g, 3: a-f, 4: a-e, 5: a-d, 6: a-c, 7: a-b, and M: marker. (C) Use of Dual-Glo luciferase to detect the inhibitory effect of targeted genes. 239FT cells were co-transfected with pll3.7-shRNA and psiCheck2 genes with different ratios, and the expression of Dual-Glo luciferase reporter genes was measured after 48 h. Data were expressed as the means ± S.E.M. (n = 3). Columns with different superscripts differ significantly, P < 0.05.

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  Figure 2. Identification of SB transposon-mediated transgenic sheep. (A) Schematic diagram of the inserted part of the pUC-VP1-shRNA expression vector, which was cut from pLL3.7 and ligated into the PUC19-IR/DR vector. (B) Pronuclear microinjection. (a): non-centrifuged fertilized ovine egg and (b): centrifuged fertilized egg (12000 × g for 5 min). (C) Electrophoresis of PCR products and (D) Southern blot analysis to identify the transgenic sheep, respectively. a: U6-VP1-shRNA group and linearized pLL3.7-VP1-shRNA plasmid group. b: IR/DR-U6-VP1-shRNA + SB100 × group. WT = wild type as a negative control; “p”: positive control; “1–8”: transgenic lambs. (E) Inhibitory effects of shRNA on VP1 gene expression in ear fibroblasts of transgenic versus wild type sheep as determined by luciferase reporter assay. Each test was repeated three times for each individual. Tg ( = transgenic sheep), N = 8; WT (wild type), N = 8. (F) A photo of the transgenic lamb. Data were expressed as the means ± S.E.M. Columns with different superscripts differed significantly, P < 0.05.

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  Figure 3. Identification of the SB transposase gene. (A) A diagraph of construction of the SB transposase gene (B) The PCR result of SB transposase gene SB100 × based on the template pCMVSB100 vector. 1: Marker, 2: SB100 × fragment.