Construction of (a) Schematic diagram of a VZV pOka genome. The 125-kb genome, VZV, contains a unique long (UL) and a unique short (US) segment. (b) Four cosmids containing overlapping VZV genomic segments are shown. A BAC vector was inserted between ORF60-61 in a VZV cosmid, pvSpe23, by homologous recombination. The BAC vector carries a GFP and a marker. (c) construction. (A) the BAC-containing cosmid was cotransfected with the three complementary cosmids into MeWo cells; (B) homologous recombination between these four cosmids forms a circular full-length VZV genome; (C) the recombinant virus replicated and produced a green plaque; (D) the circular DNA was isolated from infected cells and (E) transformed into E. coli and selected for CMR colonies; (F) the DNA was isolated from E. coli and verified by restriction digestion and partial sequencing; (G) infectivity and integrity of the were tested by transfecting BAC DNA into MeWo cells and producing VZV virus.
