Characterization of cis-Acting Sequences Involved in Canine Adenovirus Packaging  Claire Soudais, Sylvie Boutin, Eric J. Kremer  Molecular Therapy  Volume 3, Issue 4, Pages 631-640 (April 2001) DOI: 10.1006/mthe.2001.0263 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Analysis of the CAV-2 c/s-acting packaging domain. The ITR (1 to 198) is in gray. Between bp 1 and 400 there are fifteen 5'-TTTG/A-3’ motifs (underlined): six 5'-TTTG-3', nine 5'-TTTA-3’ and a single Ad5 consensus sequence containing both parts of the bipartite motif (bold and red). The probable steroid/thyroid hormone receptor binding half-sites are located in the ITR (bold and lower case). The overlapping NgoMIV and Ehel sites are in blue. P176 refers to the point mutation introduced in the Ehel site. Three approximately 30-bp gaps are located between four of the last five 5'-TTTG/A-3’ motifs (black and bold): the significance of this spacing is unknown. Molecular Therapy 2001 3, 631-640DOI: (10.1006/mthe.2001.0263) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Schematic of the construction. (a) Generation of pTCAV-1 through pTCAV-4 and the (b) CAVGFP vectors carrying mutations in the packaging domain. Numbers represent the nucleotide position relative to the CAV-2 wild type sequence. The ITR is the first 198 bp. Triangles indicate the location of the loxP sites. Deletions are indicated as open boxes. “Δ motif” refers to the motifs deleted (for numbering see Fig. 1). Mutant virus yield in single infections (yield) is expressed as fold reduction relative to CAVGFP-1. The yield following co-infection with CAVβgal (coinf) is expressed as the fold reduction based on GFP transducing units. pCAVGFP-4.3 was unrecoverable after five separate transfections. NT, not tested; NV, nonviable. Molecular Therapy 2001 3, 631-640DOI: (10.1006/mthe.2001.0263) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Analysis of CAVGFP-4 DNA isolated from DKCre and DKZeo-infected cells. (a) To the left is an ethidium bromide stained agarose gel, to the right are the results of Southern blot analysis. Restriction enzymes are denoted as E, Ecl136II; V, EcoRV; and N, NdeI. Asterisks (*) indicate the bands where the floxed sequence is located and to which the radiolabeled probe (CMV promoter) hybridized. The faint band (denoted by #) at approximately 1.9 kb in the Southern blot analysis of DKCre-infected cells may represent the linearized floxed fragment as Ecl136II cuts within the floxed sequence. M denotes the 1-kb molecular weight marker (Promega). (b) Schematic of the left end of CAVGFP-4 genome when amplified in DKZeo or DKCre cells. Shown are the relevant locations of the restriction enzyme sites used in (a). (c) Approximately 7 ml of crude CAVGFP-4 supernatant from amplification on DKCre or DKZeo cells (approximately 108 cells) was separated on a CsCl step gradient (Materials and Method). Arrow denotes the empty capsid band and mature vector particles. Molecular Therapy 2001 3, 631-640DOI: (10.1006/mthe.2001.0263) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Packaging competition assay. Cells were infected with the test vector alone or with 50 particles per cell of CAVβgal. Twofold serial dilutions of the output from this infection were used to infect a second 24-well plate. These cells were collected 24 h postinfection and the transducing unit titer (% GFP positive) was assayed by flow cytometry. Above each graph is the name of the CAVGFP vector tested. The x axis is the dilution (twofold) incubated for the second amplification. The vector amplified alone (solid line) or with CAVβgal (dashed line) was plotted as the percentage GFP-transducing units/cell. All vectors were tested in two independent assays and data shown are from one representative experiment. Molecular Therapy 2001 3, 631-640DOI: (10.1006/mthe.2001.0263) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Replication assay. DKZeo cells were infected with 50 particles per cell of the CAVGFP vector and 100 particles per cell of CAVRed (the internal control). Low-molecular-weight DNA was isolated (43) and digested with XbaI, which generated a 4.0-kb band for CAVRed and a 2.4-kb band for the test constructs harboring the GFP expression cassette. For Southern blot analysis (a) we used the CMV promoter sequence. The signal was quantified (CAVRed in black and CAVGFPs in white) and compared as the percentage of the total signal combined in the two bands (b). Molecular Therapy 2001 3, 631-640DOI: (10.1006/mthe.2001.0263) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Heat-sensitive assay. Equal aliquots of each CAVGFP vector were incubated at the indicated temperature for 15 min and then used to infect DKZeo cells. The percentage of GFP positive cells as assayed by flow cytometry is shown on the y axis. Data are means of two independent assays. Molecular Therapy 2001 3, 631-640DOI: (10.1006/mthe.2001.0263) Copyright © 2001 American Society for Gene Therapy Terms and Conditions