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Figure 1 | Veterinary Research

Figure 1

From: A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus

Figure 1

Generation and identification of the recombinant virus FA4-EGFP. A The homology-dependent knock-in strategy for generating the recombinant virus FA4-EGFP using the CRISPR/Cas 9 system. LMH cells were first transfected with sgRNA1 and sgRNA2. 24 hpt, the LMH cells were infected with FAdV-4 and transfected with donor plasmid. The recombinant virus FA4-EGFP was then purified by limiting dilution assay and viral plaque assay. B 24 hpt, the EGFP could be found by fluorescence microscopy in the transfected LMH cells with the infection of FAdV-4 at 24 hpi (a), whereas no EGFP could be observed in the FAdV-4 infected LMH cells without the transfection of sgRNA and donor plasmid (b). C PCR identification of the recombinant virus FA4-EGFP. The unpurified recombinant virus FA4-EGFP (Lane 1), the purified FA4-EGFP (Lane 2), and the WT FAdV-4 (Lane 3) were detected using specific primers listed in Table 2. D Western blot analysis of the recombinant virus FA4-EGFP. LMH cells infected with the purified recombinant virus FA4-EGFP (Lane 1) and the WT FAdV-4 (Lane 2) were harvested and lysed, and the lysates were then tested with Western blot by monoclonal antibodies against GFP.

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