1. Recombinant phenotyping.
Recombinant phenotyping. Sequential genetic constructs from top to bottom illustrate a contemporary method for determining the drug resistance phenotype of a viral mutation. Standard laboratory strain AD169 is modified with a secreted alkaline phosphatase (SEAP) reporter gene between genes US3 and US6 (strain T2211) (54). A bacterial artificial chromosome (BAC) vector is then ligated into strain T2211 DNA at this site and a full-length clone (BA1) is isolated in E. coli. The gene region to be mutagenized (UL97) is replaced with a selectable galK gene (BA9) by conditional recombination (“recombineering”) in E. coli strain SW105 (200). The desired mutant UL97 sequence is then introduced by additional recombineering, using a transfer vector containing a selectable kanamycin marker (Kan) flanked by 34-nucleotide Frt motifs. The Kan marker is subsequently removed by an inducible Flp recombinase, leaving a BAC clone (BA66) containing a specific UL97 mutation (in this case C603W) and one upstream Frt motif (41). The mutant BAC is transfected into human fibroblast cell cultures, resulting in live virus that is phenotyped for drug susceptibility using culture supernatant SEAP activity as a measure of viral growth (41, 54). (Adapted from reference 41.)‏
Nell S. Lurain, and Sunwen Chou Clin. Microbiol. Rev. 2010; doi: /CMR