DH10B E. coli containing the pOkaBAC (30 (link)) were transformed with plasmid pST76A-SR_ORF31_2096A and recA-mediated allelic exchange was carried out as described previously (71 (link)), resulting in pOkaBAC_ORF31_2096A. DH10B cells containing pOkaBAC_ORF31_2096A were transformed with plasmid pST76A-SR_ORF31_2096G, and pOkaBAC_ORF31_2096G was generated using the same procedure. pOkaBAC, pOkaBAC_ORF31_2096A, and pOkaBAC_ORF31_2096G were purified (Genopure Plasmid Maxi Kit; Roche Diagnostics), subjected to restriction fragment length polymorphism analysis using BamHI or EcoRI and the region used for allelic exchange was sequenced.
The purified BAC genome (1 μg) was mixed with PEImax solution (3 μL) prepared as described previously (79 (link)) and transfected to MRC-5 cells. After cytopathic effects were seen in cells expressing green fluorescent protein within the BAC cassette, cell-free virus was prepared as described above and used to infect MeWo_Cre cells to excise the BAC cassette using the Cre/loxP system, resulting in rpOka_gB699Q (from pOkaBAC_ORF31_2096A) and rpOka_gB699R (from pOkaBAC_ORF31_2096G).
The purified BAC genome (1 μg) was mixed with PEImax solution (3 μL) prepared as described previously (79 (link)) and transfected to MRC-5 cells. After cytopathic effects were seen in cells expressing green fluorescent protein within the BAC cassette, cell-free virus was prepared as described above and used to infect MeWo_Cre cells to excise the BAC cassette using the Cre/loxP system, resulting in rpOka_gB699Q (from pOkaBAC_ORF31_2096A) and rpOka_gB699R (from pOkaBAC_ORF31_2096G).
