*Images adapted from Dulal et al.
|
| Figure 4: VZV mutational analysis and rescue/mutant generation by galK-based mutagenesis system. A. Generation of ORF deletion mutant virus: SW102 E. coli are
made to be electrocompetent (E.C.) (1a) for VZV BAC DNA transformation (1b). Next, a galK gene is PCR amplified by primers containing 40-bp sequences homologous
to the flanking regions of the gene or region of interest (ORFX) in VZV to create the galK cassette (2a). In order to generate a deletion mutant clone (ORFXΔ), SW102
E. coli carrying WT VZV BAC undergo E.C. cell preparation with recombinase system activated (2b). The galK cassette is then electroporated into the E.C. SW102
harboring WT VZV BAC (3). Upon homologous recombination, ORFX will be replaced by the galK gene, generating the VZV ORFXΔ mutant BAC clone (4). The deletion
clone is then verified by PCR and sensitivity to antibiotics, and extracted via BAC Maxipreparation (5). The mutant BAC is then chemically transfected into human
ARPE-19 cells (6) and grown for mutant analysis (7). B. Generation of ORF rescue/mutant VZV virus: SW102 E. coli harboring VZVLUC ORFXΔ mutant BAC is made
electrocompetent and recombination system activated (1a). ORFX (for rescue generation) or ORFY (any other gene, or mutated ORFX) is PCR amplified with primers
conferring homology to flanking regions in ORFXΔ mutant BAC (1b), so that upon transformation (2) and homologous recombination (3), the ORFX/Y cassette will
replace the galK gene in the ORFXΔ mutant BAC. After PCR verification, growth property confirmation, and extraction of the rescue/mutant BAC via Maxi preparation
(4), the ORFX Rescue/ORFY Mutant BAC is chemically transfected into human ARPE-19 cells (5). The virus that results is then grown and analyzed for comparison to
WT VZV (6).
*Images adapted from Dulal et al. |
