Cre recombinase

One acceptor and one or multiple donor vectors were assembled using Cre-mediated recombination as previously described (33 (link),38–39 (link)). One acceptor and one or more donors were mixed with a ratio of 1:1.1 in distilled H2O with 0.5 ul (7.5 U) of CRE recombinase (NEB # M0298M) and 1 ul Cre buffer (provided with CRE recombinase) to a final volume of 10 ul in distilled H₂O. 500–1000 ng of total DNA were used for each reaction. Cre-reactions were incubated for 1 hour at 37°C, followed by heat inactivation at 70°C for 10 min. 2–3 ul were transformed into homemade electrocompetent Top10 E. coli, followed by 2 h recovery at 37°C and plating on LB/agar plates with the appropriate antibiotics. Cre-recombination products were predicted using Cre-ACEMBLER Vers. 2.0 (38 (link)).

PRV DX viral DNA was extracted as mentioned earlier and the transfer plasmid pUC18-gE-loxp-cherry (3 μg) was co-transfected with the genomic DNA of PRV DX (1 μg) into Vero cells for homologous recombination. After six times of plaque purification, the gE-deleted virus expressing cherry was generated and named as PRV DX-gE-loxp-cherry. Afterward, the genomic DNA of PRV DX-ΔgE-cherry was extracted and dealt with cre recombinase (New England Biolabs, USA), then the viral DNA was extracted and transfected into Vero cells. Undergoing plaque purification for six times, PRV gE-deleted virus was generated and named as PRV DXΔgE strain. Then 3 μg of pUC18-TK-loxp-EGFP plasmid and 1 μg of PRV DXΔgE genome DNA were co-transfected into Vero cells. Plaques with green fluorescence were purified by six rounds, viral DNA was extracted and excised with cre recombinase and then transfected into Vero cells as described earlier. After another plaque purification for six times, the gE/TK-deleted PRV (namely, HD/c) was generated, and the genome were confirmed by sequencing (Biosune, China).