Precise targeted integration by a chimaeric transposase zinc-finger fusion protein

Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inapp...

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Bibliographic Details
Published in:Nucleic acids research 2010-03, Vol.38 (4), p.1204-1216
Main Authors: Feng, Xiaofeng, Bednarz, Amy L, Colloms, Sean D
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Early Growth Response Protein 1 - chemistry
Early Growth Response Protein 1 - genetics
Early Growth Response Protein 1 - metabolism
Gene Targeting
Human immunodeficiency virus 1
Mice
Molecular Biology
Plasmids - genetics
Recombinant Fusion Proteins - metabolism
Transposases - genetics
Transposases - metabolism
Zinc Fingers
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Summary:Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer 'Z-transposases' that could deliver transgenic cargoes to chosen genomic locations.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkp1068