Site-specific PCR for detection of targeted transgene integration.

A. A representative image of results; lentivirus vectors marked above lanes (NTD = non-transduced control). The samples are flanked by a molecular weight marker (MassRuler DNA Ladder Mix, Thermo Scientific SM0403) and relevant marker bands are annotated on the right. Arrows show expected product sizes from targeted transgene integration; blue arrows show homologous recombination (HR) and non-homologous end-joining (NHEJ) products expected from LVs containing rDNA-compatible homology arms (HA), green arrow shows NHEJ product expected from LVs without HA. B. A representative alignment result showing the attachment of the D+H vector’s LTR to the genomic DNA. A product of the site-specific PCR (A) was sequenced and aligned with a template sequence constructed to model the NHEJ-insertion of a vector genome into the I-PpoI cleavage site. The GT-dinucleotide (green annotation) is present at the end of the vector’s 3’ LTR sequence indicative of transgene insertion via NHEJ. (TIF)