Site-directed transposon integration in human cells.
The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.
Yant
Stephen R
SR
Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
Huang
Yong
Y
Akache
Bassel
B
Kay
Mark A
MA
D001665:Binding Sites; D002384:Catalysis; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000737:chemistry; D018014:Gene Transfer Techniques; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D019895:Transposases / Q000235:genetics* / Q000378:metabolism*
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019895:Transposases; C494471:sleeping beauty transposase, human