Targeted knockout of Mx in the DF-1 chicken fibroblast cell line impairs immune response against Newcastle disease virus

Figures

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  Figure 1. Establishment of chicken Mx knockout cell lines using the CRISPR/Cas9 system. (A) Structure of the Mx and location of the target1 and target2 sites. (B) The constructed CRISPR/Cas9 vectors were validated by Sanger sequencing. (C) T7E1 assay was performed in DF-1 cells infected with the Mx-sgRNA1 and Mx-sgRNA2 recombinant viruses. (D) Sequencing analysis of Mx knockout events for single cell clones. The red letters indicate guide RNA binding sites. (E) Detect the expression of Mx in DF-1 cells post-NDV infection via Western-blot (**P < 0.01). (F) The viability of Mx−/− DF-1 cells.

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  Figure 2. Mx knockout impaired DF-1 cells anti-NDV ability. (A) NDV infection causes lesions of DF-1 cells. The solid lines are normal cells, and the dotted lines are diseased cells. (B) The expression level of Mx was detected by western blot (**P < 0.01). (C) RT-qPCR was used to detect the NDV copy numbers (**P < 0.01).

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  Figure 3. Antiviral response in Mx−/− cell lines post-NDV infection. (A–I) The relative expressions of IRF-7, IFN-α, IFN-β were assessed by RT-qPCR. (J–L) The relative expression of TNF-α was assessed by RT-qPCR (**P < 0.01).

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  Figure 4. Effect of Mx knockout on the expression of cyclin-dependent kinase in chicken DF-1 cells post-NDV infection. (A–F) The relative expressions of cyclin-dependent kinase p21, p27 were assessed by RT-qPCR (*P < 0.05, ** P < 0.01).

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  Figure 5. Mx deletion promoted cell apoptosis. (A–C) The relative expression of pro-apoptotic factor Bak was assessed by RT-qPCR, (**P < 0.01). (D) Cell apoptosis was determined by TUNEL assay in Mx−/− DF-1 cells.