Table 2.

Yield of circular intermediates isolated from Hirt DNA

Bacterial transformationStarting no. of plasmid or AAV genome moleculesActual no. of ampicillin-resistant CFUAdjusted yield (CFU) for 3 × 1010 molecules
Hirt DNA from rAAV-infected musclea 3 × 1010 5 × 103 5 × 105e
Hirt DNA + 10 ng of LacZ plasmidb 1.3 × 109 8.7 × 104c 2 × 108d
10 ng of LacZ plasmid1.3 × 109 8.7 × 106 2 × 108d
  • a The actual amount of Hirt DNA used for transformation was 3/20 the entire Hirt DNA. The numbers have been adjusted to reflect viral inoculum and yields for the entire muscle.

  • b Plasmid DNA was spiked into mock-infected muscle homogenates prior to isolation of Hirt DNA. This reconstituted Hirt DNA was then used for transformation of bacteria.

  • c The average of several experiments indicates an approximate 100-fold reduction in the number of CFU recovered from bacterial transformations with DNA isolated from Hirt extract spiked with plasmids compared to transformation with an equivalent amount of plasmid DNA alone.

  • d The number of CFU in plasmid reconstitution studies have been adjusted to normalize the number of plasmids genomes to that used in AAV experiments (3 × 1010). Control LacZ plasmid was approximately 7,000 bp with a molecular size of 4.6 × 106 g/mol. Therefore, 230 ng of plasmid is equivalent to 3 × 1010 molecules (i.e., normalization factor of 23-fold).

  • e Adjusted yield indicates that approximately 1 in 400 AAV genomes circularizes in vivo.