Figure 5. Testing the inducible rep68 expression unit in dHV.68/5939.F50 particles purified by CsCl density gradient ultracentrifugation. (A) Western blot analysis of FLPe-induced Rep68 synthesis. HeLa cells were co-transduced with dHV.F50 and hcAd. FLPe.F50 (lane 1) or with dHV.68/5939.F50 and hcAd.FLPe.F50 (lane 3) or were transduced exclusively with dHV.F50 (lane 2) or with dHV.68/ 5939.F50 (lane 4). Three days postinfection, cellular proteins were extracted, separated in an SDS-10% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Next, the membrane was incubated with a monoclonal antibody specific for all four AAV rep gene products. An antibody directed against actin served as a loading control. (B) eGFP direct fluorescence microscopy and Rep68 immuno- fluorescence microscopy on HeLa cells infected with hcAd.FLPe.F50 (50 GSAUs/cell) and either dHV.F50 (5 HTUs/cell; left column) or dHV.68/5939.F50 (5 HTUs/cell; right column). The pictures are derived from representative microscopic fields. The fluorescent signals derived from the green and red channels are overlaid in the lower panels. The arrowheads in the right panels point to cells containing both eGFP and Rep68. Note that the former protein is distributed throughout the cell whereas the latter is confined to the nucleus. Original magnification: 4006. doi:10.1371/journal.pone.0003084.g005 specific probe (Fig. 6A; upper panel) and, after membrane stripping, was exposed to a mixture of two probes binding immediately downstream of the rep68 gene and close to the right end of the monomeric vector genome (Fig. 6A; lower panel). Examples of autoradiograms derived from these experiments are shown in Fig. 6B (HeLa cell clones) and Fig. 6C (DMD myoblast clones). Hybridization of a particular Eco32I fragment with both the AAVS1- and dHV.68/5939.F50-specific probes signifies inser- Targeted Large DNA Insertion tion of vector DNA into the AAVS1 locus on human chromosome 19 (Figs. 6B and 6C, open arrowheads). Subsequently, the membrane was once again stripped and incubated with a probe specific for a 1.9-kb Eco32I fragment in the vector genome (Fig. 6A; lower panel). The autoradiograms corresponding to hybridizations using the AAVS1-specific probe showed that 50% of the 38 randomly selected HeLa cell clones yielded DNA fragments with larger and, less often, smaller sizes than that of the 5.6-kb Eco32I fragment characteristic for undisrupted AAVS1 alleles (Fig. 6B; upper panel and Table 1). This observation is consistent with the well-known capacity of Rep78 and/or Rep68 to locally amplify the AAVS1 region due to the usage of its RBE and trs as an origin of replication [33]. Thus, the absence of detectable changes to the AAVS1 loci in the other 50% of the HeLa cell clones most likely reflects the presence of no or few Rep68 molecules in their founder cells probably due to a lack of FLPe. In agreement with this interpretation, no disruptions of the AAVS1 alleles were found after Southern blot analysis of chromosomal DNA extracted from 40 randomly selected clones of HeLa cells exposed exclusively to dHV.68/5939.F50 (Fig. 6D; Table 1), indicating that although ‘‘leaky’’ rep68 expression may occur (Fig. 5A) in most cases the amount of Rep68 does not suffice to amplify the AAVS1 region. Consistent with this finding, Southern blot hybridization using the vector-specific probes revealed that these clones did not have dHV.68/5939.F50 DNA integrated into the AAVS1 region (data not shown). Conversely, in the presence of the FLPe-encoding hcAd vector, integration of dHV.68/5939.F50 DNA into the AAVS1 locus could be demonstrated (Fig. 6B; middle panel and Table 1). The targeted DNA integration efficiency, as determined on the basis of the total number of HeLa cell clones analyzed was 24% (9/38), whereas if one considers exclusively clones with discernible changes in the AAVS1 locus (as a surrogate indicator for cells that originally contained enough Rep68) it reached 47% (9/ 19; Fig. 6B; Table 1). The same analysis applied to autoradio- grams corresponding to DMD myoblast clones revealed that these cells were targeted at the AAVS1 locus with an efficient of 28% (5/ 18) or 100% (5/5; Fig. 6C; Table 1), respectively. Molecular characterization of AAVS1-foreign DNA junctions following dHV.68/5939.F50-mediated gene transfer To confirm by an independent method the ability of rep68- positive dHV particles to accomplish locus-specific foreign DNA insertion, we performed experiments aiming at the isolation of junctions between AAVS1 and vector DNA. To this end, DMD myoblasts were transduced with dHV.68/5939.F50 alone or together with hcAd.FLPe.F50. Subsequently, chromosomal DNA was extracted from the transduced cells and AAVS1-vector DNA junctions were analyzed by PCR (Fig. 7A). Chromosomal DNA derived from human cells stably transduced by dHV.68/5939.F50 DNA in a non-targeted (i.e. HeLa cell clone N+2) and AAVS1- specific (i.e. DMD myoblast clone 3C5) manner served as negative and positive controls, respectively. The DNA derived from the DMD myoblast clone yielded a discrete PCR fragment whereas no amplification product was obtained with the DNA from the HeLa cell clone (Fig. 7B, lower panel, compare lanes 1 and 2). Nested PCR products were also detected in samples corresponding to reactions performed on DNA from DMD myoblast cultures transduced with dHV.68/5939.F50 alone (Fig. 7B, lower panel, lane 3) or together with hcAd.FLPe.F50 (Fig. 7B, lower panel, lane 4). Importantly, however, a much broader range of PCR product sizes was present in the sample derived from the cells that had been exposed to both vectors (Fig. 7B, lower panel, compare lanes 3 and 4). These results indicate dHV.68/5939.F50 DNA PLoS ONE | www.plosone.org 7 August 2008 | Volume 3 | Issue 8 | e3084