Screening of bovine
shRNA and construction of its eukaryotic expression vector. A. Effect of designed bovine CD14 shRNAs was detected by qRT-PCR analysis. The lentiviral particles expressing CD14 shRNAs were used to infect HEK 293 cells expressing bovine CD14, non-infected cell line as a blank control, the scrambled shRNA as negative control. The values for columns with different letters represent statistically significant differences, p < 0.01. B. The inhibition effect of CD14 shRNA-674 fragment was confirmed by western blot analysis. HEK 293 cells stably expressing bovine CD14 were infected by shRNA-674 lentivirus at two different MOIs (lane1, 2), the negative control was HEK 293 cells stably expressing bovine CD14 (lane 3). C. Identification of the pSilencer™4.1-CD14-IRES recombinant plasmid. M: 1 kb Marker; Lane1: pSilencer™4.1-CD14shRNA-IRES plasmid: Lane2: pSilencer™4.1-CD14shRNA-IRES plasmid digested by SspI enzyme; Lane3: pSilencer™4.1-CD14shRNA-IRES plasmid digested by HpaI and BamHI enzyme. D. Map of pSilencer™4.1-CD14 shRNA-IRES vector. E. Confirmation of F1 generation transgenic mice by Southern blot analysis. Three F1 mice were selected, among them, two (offspring of 22#, 32#) were transgenic while the third was not (offspring of 11#) by RT-PCR analysis. The positive control was pSilencer™4.1-CD14 shRNA-674-IRES plasmid. M: Marker; Lane 1: positive control; Lane 2: offspring of 22# mouse; Lane 3: offspring of 11# mouse; Lane 4: offspring of 32# mouse.