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Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):E2279-87. doi: 10.1073/pnas.1305987110. Epub 2013 May 30.

piggyBac transposase tools for genome engineering.

Author information

1
Howard Hughes Medical Institute and Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Abstract

The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc(+)Int(-)) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc(+)Int(-) transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int(-) phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc(+)Int(-) transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.

KEYWORDS:

GULOP; ROSA26; induced pluripotent stem cell production; protein-DNA interaction

PMID:
23723351
PMCID:
PMC3690869
DOI:
10.1073/pnas.1305987110
[Indexed for MEDLINE]
Free PMC Article
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