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

Feng, X., Bednarz, A.L. and Colloms, S.D. (2010) Precise targeted integration by a chimaeric transposase zinc-finger fusion protein. Nucleic Acids Research, 38(4), pp. 1204-1216. (doi: 10.1093/nar/gkp1068)

Abstract

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Bednarz, Dr Amy and Colloms, Dr Sean
• Authors: Feng, X., Bednarz, A.L., and Colloms, S.D.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry; Q Science > QH Natural history > QH426 Genetics
• College/School: College of Medical Veterinary and Life Sciences
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Published Online: 03 December 2009
• Copyright Holders: Copyright © 2009 The Authors.
• First Published: First published in Nucleic Acids Research 38(4):1204-1216
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher.

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
25233	1	Interactions between Proteins Bound at Distant Sites on DNA Molecules: Involvement in Site-Specific Recombination ...	Sean Colloms	Wellcome Trust (WELLCOME)	057651/Z/99/CH/	Institute of Molecular Cell and Systems Biology
25233	2	Interactions between Proteins Bound at Distant Sites on DNA Molecules: Involvement in Site-Specific Recombination ...	Sean Colloms	Wellcome Trust (WELLCOME)	57651	Institute of Molecular Cell and Systems Biology
25233	3	Interactions between Proteins Bound at Distant Sites on DNA Molecules: Involvement in Site-Specific Recombination ...	Sean Colloms	Wellcome Trust (WELLCOME)	057651/Z/99/A	Institute of Molecular Cell and Systems Biology
31176	1	Transposition of IS630: towards a site-specific integration vector for genetic engineering (Mr.X. Feng)	Sean Colloms	Wellcome Trust (WELLCOME)	066001/Z/01/Z/K	Institute of Molecular Cell and Systems Biology
53731	1	Wellcome Open Access Award	Sean Colloms	Wellcome Trust (WELLCOME)	090726/Z/09/Z	Institute of Molecular Cell and Systems Biology