Journal: The Journal of Clinical Investigation
Article Title: Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice
Figure Lengend Snippet: PCR identification of DNA flanking the SB -Tn insertion sites. ( A ) DNA isolated from the livers of hemophilia A mice injected with HA pT2/CAGGS-BΔcFVIII//IF SB 10 nanocapsules and wild-type controls served as template using primer pairs specific for the FVIII hemophilia A knockout (top) or the wild-type allele (bottom). ( B ) Lack of SB 10 CDS persistence in the HA-treated knockout mice. PCR amplification of the SB ) using liver DNA isolated from the DsRed2 animals and treated hemophilia A mice. The size or identity of the predicted amplicons and selected bands of the DNA marker (M) are indicated. The time (after injection) that the livers were harvested is indicated above the gels. ( C ) Schematic of the inverted-nested PCR strategy used to identify DNA flanking the SB -Tn insertion site. The genomic DNA digested by NcoI or XhoI was subjected to self-ligation and the products used as template for the initial inverted PCR amplification with primer pairs RP1/LP1 and RNP1/FNP1 for XhoI- and NcoI-digested DNA, respectively. The second PCR amplification used the XhoI and NcoI initial reactions as template with internal nested primer pairs LP2/RP2 and RNP2/FNP2, respectively. The PCR products were analyzed by agarose gel electrophoresis and visualized by UV light after ethidium bromide staining. The size of the 3 heavy bands is shown to the left of the NcoI gel. ( D ) Identification of the insertion sites in hemophilia A mice treated with HA-encapsulated pT2/CAGGS-BΔcFVIII//IF SB 10. The region of the ID/DR of the Tn and the requisite duplicated TA (gray) followed by 40 nt of genomic DNA flanking the identified insertion sites are indicated above the sequences. The chromosomal location established by BLAST analysis is shown at right, and the intronic insertion sites are marked with asterisks. The closest adjacent genes for the other insertions are listed, with the distance in kb from the Tn insertion indicated in parentheses if less than 100 kb.
Article Snippet: The mouse eukaryotic initiation factor 4A1 promoter–driven (IF-driven) SB 10 and the pT2/IF SB 10 were generated by blunt-end cloning of SB 10 CDS into the NcoI/EcoRI-digested pDrive-meIF4A1 vector (InvivoGen).
Techniques: Polymerase Chain Reaction, Isolation, Mouse Assay, Injection, Knock-Out, Amplification, Marker, Nested PCR, Ligation, Agarose Gel Electrophoresis, Staining