Clonase

The serotype 5 adenovectors used in this work contain the RGD modification in the fiber protein [42] (link). The backbone provided by Dr. Hiroyuki Mizuguchi (Osaka University, Japan) was modified to allow in vitro recombination using clonase (Invitrogen, 12538120) (A.H., manuscript submitted). Adenovectors express eGFP, IFNβ, and p19Arf under the control of a p53 responsive promoter [43] (link) or LacZ the under control of the CMV promoter.
Adenovector production was performed by transfection of linearized plasmids into HEK293A cells followed by amplification cycles and purification using iodixanol gradient [44] (link). Purified adenovectors were stored in PBS 7% glycerol at −80°C. Titration was done using Adeno-X Rapid Titer Kit (Clontech, 632250).

In-frame deletions of selected genes were constructed by allelic replacement using pKOR1 (46 (link)). Primers X1-X2 (see Table S3 in the supplemental material) were used to amplify approximately 1,000 bp that corresponded to the first 84 to 153 nucleotides from the start codon and flanking 5′ region. Primers X3-X4 were used to amplify approximately 1,000 bp that corresponded to the last 33 to 159 nucleotides from the stop codon and flanking 3′ region (see Table S3). X1-X2 and X3-X4 PCR products were spliced together by overlap PCR using primers X5 and X6. Attachment sites (attB), appended to 5′ ends of primers X5 and X6 were recombined with the attP sequences flanking a lambda recombination cassette on pKOR1 in the presence of bacteriophage lambda integrase and Escherichia coli integration host factor (Clonase; Invitrogen) and electroporated into E. coli. The in-frame deletion constructs were electroporated into S. aureus RN4220 and then transduced with Φ11 into SF8300. Allelic replacement was performed as described elsewhere (46 (link)). Allelic replacement mutants were identified by PCR and DNA sequencing using primers X1 to X4 and primers X1, X4, S1, and S2, respectively (see Table S3). Multiple gene deletions were carried out sequentially.