Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells
Schematic diagram of the internally controlled pKT2/NAG vector and integration products mediated by Sleeping Beauty (SB) transposase and by bacteriophage PhiC31 (C31) integrase. (a) The integrating vector consists of (i) a C31 integrase recognition site (attB); (ii) eF1α or CMV- (Pr-) regulated GFP expression unit; (iii) neomycin resistance gene (Neo) transcriptionally regulated by the PGK promoter; (iv) flanking T2 transposase binding sites (IR/DRs; boxes with double triangles) separated by; (v) a colE1 bacterial origin of replication and kanamycin resistance gene; (vi) pA, polyadenylation signal from the rabbit beta globin gene. (b) SB transposase-mediated integration (left): the SB transposase excises transposon sequences at IR/DR transposase binding sites and precisely inserts them into TA-dinucleotide targets in cellular chromosomes, which are subsequently duplicated. C31 integrase-mediated integration (right): exogenous gene sequences on an attB containing plasmid integrate into mammalian genomes at “pseudo-attP” sites, chromosomal sequences having partial homology to the wild-type phage attP sequence.
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