Construction of (a) HCMV genome and bacterial plasmid with BAC vector that has been linearized and transfected into human fibroblast cells. (b) BAC vector is inserted into HCMV genome by homologous recombination. The viral genome (now containing the BAC vector) will naturally circularize during replication. (c) HCMV viral BACs are selected based upon expression of a GFP cassette within the BAC vector. (d) Fluorescent plaques are isolated, and viral BAC DNA is extracted. (e) Viral BACs inserted into E. coli cells via electroporation. Successful integration of the BAC vector into the viral genome can be confirmed by () selecting colonies with antibiotic resistance resulting in the chloramphenicol-selectable marker in the BAC vector and () confirming the BAC genome sequence has not gained any undesired mutations by using restriction digest analysis to compare the BAC DNA to the original viral DNA. (f) If desired, mutagenize the HCMV viral BAC (via site-directed mutagenesis or random transposon mutagenesis). Either way, viral BAC DNA must be isolated via Maxiprep for transfection back into human cells. (g) Transfect BAC DNA into human fibroblast cells in order to produce infectious virus.
