Figure 1.
Overview of the poxvirus Selectable and Excisable Marker cloning system.
(A) Schematic illustrating the Selectable and Excisable Marker poxvirus cloning system. (B) First generation poxvirus cloning vector pSEM-1 with labeled open reading frames. (C) Immunofluorescence detection of Cre recombinase from U2OS cell lines expressing nuclear or cytoplasmic Cre, or control U2OS cells. (D) Western blot analysis of Cre recombinase from U2OS cells mock transfected (U2OS), transiently transfected (pMC-Cre) or stably expressing Cre recombinase targeted to either the nucleus or the cytoplasm (Nuc-Cre and Cyto-Cre respectively).
Figure 2.
FACS purification of recombinant VV-ΔTk infected cells and Cre-recombinase mediated removal of the selectable marker.
(A) Dot plot of YFP fluorescence versus side scatter from Fluorescence Activated Cell Sorting (FACS) analysis of U2OS cells mock infected, or infected with a mixture of parental VV and recombinant VV virus expressing YFP. (B) Purified recombinant VV-ΔTk-yfp-gpt-1loxP (control virus) or VV-ΔTk-yfp-gpt virus were used to infect stable cytoplasmic Cre-expressing cells (U2OS-Cre) or parental U2OS cells in 6-well plates, and were monitored for foreign gene expression (firefly luciferase) and marker gene expression (YFP-GPT fusion protein). (C) Percent YFP-negative VV-ΔTk' plaques on U2OS cells after passage of virus on Cre cells (nuclear or cytoplasmic stable cell lines or transiently transfected U2OS cells). (D) Map of the pSEM-1 plasmid indicating the primer pairs used in the PCR reactions to characterize the genome of recombinant VV-ΔTk viruses shown in panel E. (E) PCR analysis of DNA extracted from VV-ΔTk-yfp-gpt-1loxP (control virus), VV-ΔTk-yfp-gpt and VV-ΔTk' viruses. The PCR products span: i. across the left Tk flanking region, ii. across the luciferase gene, iii. across the yfp-gpt selectable marker, iv. across the right the Tk flanking region. n.s = not-significant, * p = 0.001.
Figure 3.
Confirmation of genomic composition of 3 independent recombinant VV-ΔTk viruses.
(A) An ethidium bromide stained DNA gel of genomic HindIII restriction digests of viral DNA isolated from parental VV (Wyeth Strain), 3 clones of VV-ΔTk-yfp-gpt and 3 clones of VV-ΔTk'. Arrows indicate the Tk insertion site (VV-Wyeth), and the unique bands that result from insertion of the yfp-gpt cassette (VV-ΔTk-yfp-gpt). (B) Southern hybridization of the DNA gel in A identifying the yfp insert present in the genome of the VV-ΔTk-yfp-gpt clones, but not in parental VV-Wyeth or the VV-ΔTk' clones. (C) DNAStar sequence alignment at the yfp-gpt insertion site of DNA isolated from the 3 VV-ΔTk' clones post Cre passage.
Figure 4.
Creation of the VV-ΔI4L mutants from the second generation SEM cloning vectors.
(A) Map of cloning vector pDGloxPKODEL-ΔI4L and (B) pDGloxPKOINV-ΔI4L with labeled open reading frames. (C) Schematic displaying the strategy for knock-out of the I4L open reading frame from VV strain WR, and possible outcomes of Cre-recombination of recombinant VV-ΔI4L virus generated from either the pDGloxPKODEL-ΔI4L or pDGloxPKOINV-ΔI4L vectors. (D) DNA sequence analysis of the VV-ΔI4LDEL virus post Cre passage.
Figure 5.
Cre-mediated recombination of vaccinia virus DNA is dependent upon loxP site orientation.
(A) PCR analysis of the I4L locus using primers flanking the yfp-gpt cassette of two purified ΔI4L strains generated from either the pDGloxPKODEL-ΔI4L or pDGloxPKOINV-ΔI4L vectors before (BSC-40) and after (U2OS-Cre) Cre-recombination. (B) PCR analysis of two purified ΔI4L strains generated from the pDGloxPKOINV-ΔI4L vector with primers amplifying inside and outside the yfp-gpt cassette before (BSC-40) and after (U20S-Cre) Cre-recombination. (C) Western blot analysis of two purified ΔI4L strains generated from either the pDGloxPKODEL-ΔI4L or pDGloxPKOINV-ΔI4L vectors for I4, YFP, I3 (positive control for infection) and actin before (BSC-40) and after (U20S-Cre) Cre-recombination.
Figure 6.
Generation of a recombinant ECTV using SEM.
(A) Map of pDGloxPKODEL-ΔEVM002 with labeled open reading frames. (B) Schematic displaying the strategy for knock-out of the EVM002 open reading frame from ECTV. (C) PCR analysis of viral DNA. Primers were used to amplify the regions of pDGloxPKO-ΔEVM002 homology from wild-type ECTV (ECTV-wt), ECTV-ΔEVM002-yfp-gpt, and ECTV-ΔEVM002'. (D) DNA sequence analysis of the ECTV-ΔEVM002' virus (post Cre passage). (E) Confocal microscopy was used to detect YFP fluorescence in mock infected BGMK cells or BGMK cells infected with ECTV-wt, ECTV-ΔEVM002-yfp-gpt, or ECTV-ΔEVM002'. A 40× magnification lens was used to detect differential interference contrast (DIC), and YFP fluorescence.