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linear amplification PCR (LAM-PCR) followed by sequence analyses
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To confirm editing at the molecular level, we employed linear amplification PCR (LAM-PCR) followed by sequence analyses. LAM-PCR was initiated in the chromosomal sequences external to the HAs, spanning both HAs and reading into the luciferase ORF. Sequence analyses confirmed accurate insertion into the intended location, intron 1 of the Rosa26 gene (Fig. 5C). No indel mutations or AAV ITRs were detected in the edited genomes. We further confirmed TI of the luciferase cassette into the Rosa26 locus by Southern blot analysis of mouse liver DNA, harvested 70 d after injection of AAVHSC15 Rosa26-Luc. The expected 7-kb band was visualized following hybridization of SpeI-digested DNA with a luciferase-specific probe (Fig. 5D). No luciferase-specific bands were detectable in untreated liver DNA from control mice (Fig. 5D), showing specificity. Interestingly, no 3,966-bp monomer-length free vector genomes were detected in the Southern blot analysis, possibly suggesting that the editing genome may either not have initiated second-strand synthesis or not formed double-stranded concatamers commonly observed following AAV transduction (70). Digestion of double-stranded concatamers with a single cutter enzyme would be expected to yield monomer-length AAV vector genomes, which were not observed, suggesting that such concatamers were absent. If any single-stranded AAV genomes were present, they would likely have formed “snapback” structures due to the complementarity of the ITRs and may have migrated faster in nondenaturing gels. If free vector genomes persisted as double-stranded monomers, SpeI digestion would yield fragments of 2,783 bp and 1,183 bp (Fig. 5D), which were not observed. It is also possible that the number of free vector genomes was below the limit of detection for the Southern blot assay (71). However, the presence of a 7-kb band on the Southern blot analysis, together with in vivo expression and sequence analysis, confirmed the accurate targeted insertion of the luciferase ORF into intron 1 of Rosa26 following a single i.v. injection of the AAVHSC15 editing vector. Southern blot analysis also revealed no discernible off-target bands, suggesting the absence of off-target integrations within the limits of detection of the assay.
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