Site-directed transposon integration in human cells.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Yant SR,Huang Y,Akache B,Kay MA

doi

10.1093/nar/gkm089

subject

Has Abstract

pub_date

2007-01-01 00:00:00

pages

e50

issue

7

eissn

0305-1048

issn

1362-4962

pii

gkm089

journal_volume

35

pub_type

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