Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells
Figure 3
Molecular characterization of the Sleeping Beauty-mediated integration events in GABEB cell clones.
(A) Southern blot analysis of genomic DNA from GABEB cell clones digested with NheI (SA clones) or AflII (T2 clones), single cutter in the transposon cassette, and hybridized to a Venus probe. A single band higher than 4.2 kb (SA clones) and 3.4 kb (T2 clones) indicates integration of one copy of the transposon into the genome. Multiple Venus-specific bands correspond to repeated integration events. (B) Southern Blot analysis of genomic DNA from 8 (SA) and 9 (T2) clones digested with NcoI (SA clones) or MfeI and NdeI (T2 clones). The expected Venus-specific band corresponding to 6 kb for SA and 8.9 kb for T2 transposon indicates the correct integration of the transposons into the genome. C, mock-transfected cells; red bars, Venus-specific probe. Clone showing rearrangement of the transposon cassette is highlighted by black asterisk. (C) Bi-directional mapping of the junctions between transposon and genomic DNA. The table summarizes 27 integrations belonging to 10 single clones. For each integrant, the underlined sequence represents a portion of the transposon IRs, left (CAGTT) and right (AACTG) separated by dots; TA dinucleotide (in bold) is the target site correctly duplicated after transposition. Hit chromosomes and positions are reported. UnK, unknown region of the human genome based on UCSC hg19 assembly.
