Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
PLoS One. 2012;7(11):e42666. doi: 10.1371/journal.pone.0042666. Epub 2012 Nov 15.

Comparative analysis of the recently discovered hAT transposon TcBuster in human cells.

Author information

  • 1Department of Medicine, Division of Nephrology, Baylor College of Medicine, Houston, Texas, USA.

Abstract

BACKGROUND:

Transposons are useful tools for creating transgenic organisms, insertional mutagenesis, and genome engineering. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes.

METHODOLOGY/PRINCIPAL FINDINGS:

We tested TcBuster for its activity in human embryonic kidney 293 (HEK-293) cells. Excision footprints obtained from HEK-293 cells contained small insertions and deletions consistent with a hAT-type repair mechanism of hairpin formation and non-homologous end-joining. Genome-wide analysis of 23,417 piggyBac, 30,303 Sleeping Beauty, and 27,985 TcBuster integrations in HEK-293 cells revealed a uniquely different integration pattern when compared to other transposon systems with regards to genomic elements. TcBuster experimental conditions were optimized to assay TcBuster activity in HEK-293 cells by colony assay selection for a neomycin-containing transposon. Increasing transposon plasmid increased the number of colonies, whereas gene transfer activity dependent on codon-optimized transposase plasmid peaked at 100 ng with decreased colonies at the highest doses of transposase DNA. Expression of the related human proteins Buster1, Buster3, and SCAND3 in HEK-293 cells did not result in genomic integration of the TcBuster transposon. TcBuster, Tol2, and piggyBac were compared directly at different ratios of transposon to transposase and found to be approximately comparable while having their own ratio preferences.

CONCLUSIONS/SIGNIFICANCE:

TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.

PMID:
23166581
[PubMed - indexed for MEDLINE]
PMCID:
PMC3499496
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Public Library of Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk