Table 1.

Circular AAV genomes rescued from rAAV-infected muscle

Vector	Viral dose*	E1-positive cfu†	E1/E2-positive cfu†	% E1-positive also E2-positive‡	% E1/E2-positive also Ampr/Kanr	%§Ampr also Kanr	KanrAmpr head-to-tail¶	KanrAmpr head-to-head¶
E1/E2	6  × 1010	8,671  ± 483	3,208  ± 148	36  ± 3.1	100	29  ± 2.8	10/36	15/36
E1	6  × 1010	10,956  ± 401	0	0	0	0	—	—
E2	6  × 1010	0	0	0	0	0	—	—
E1/E2	4  × 1011	27,159  ± 6,031	11,317  ± 3,398	37  ± 4.8	100	32  ± 4.4	16/56	9/56
E1	4  × 1011	26,800  ± 713	0	0	0	0	—	—
E2	4  × 1011	0	0	0	0	0	—	—

^*Samples were harvested at 110 days (low dose) and 150 days (high dose), and three muscle samples were analyzed for each experimental condition. 

^† Hirt DNA was prepared, and Amp^r colonies were rescued by transformation into bacteria. Colony hybridization was performed against DNA probes from regions of AV.Epo1 (E1) and AV.Epo1 (E2) on 100 Amp^r colonies from each experimental sample point. The total colony-forming units (cfu) from each muscle sample were then calculated from the percentage of E1 and/or E2 hybridizing colonies and the total cfu count (mean ± SEM, n = 6). 

^‡ Calculated from paired values of individual data points (mean ± SEM, n = 6). 

^§ Amp^r/Kan^r colonies were determined by replica plating of 100 Amp^r colonies onto kanamycin-containing media from each time point (mean ± SEM, n = 6). 

^¶ Numbers of head-to-tail or head-to-head genomes were determined by Southern blot analysis of Kan^rAmp^r-rescued clones from Hirt DNA. Twenty-four of the 26 plasmids with evidence of head-to-tail conformation expressed both Epo and EGFP when transiently transfected into HeLa cells. None of the head-to-head genomes expressed Epo or EGFP when transfected into HeLa cells. The remaining rescued circular intermediates had either mixed (head-to-tail and head-to-head) orientations consistent with large multimer concatamers or structures not easily identifiable with the present restriction enzymes used [i.e., could contain small deletions or rearrangements as previously reported for large multimer circular concatamers (10)].