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A randomized library approach to identifying functional lox site domains for the Cre recombinase
The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about...
Autores principales: | , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018622/ https://www.ncbi.nlm.nih.gov/pubmed/17702764 http://dx.doi.org/10.1093/nar/gkm604 |
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author | Sheren, Jamie Langer, Stephen J. Leinwand, Leslie A. |
author_facet | Sheren, Jamie Langer, Stephen J. Leinwand, Leslie A. |
author_sort | Sheren, Jamie |
collection | PubMed |
description | The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system. |
format | Text |
id | pubmed-2018622 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-20186222007-10-23 A randomized library approach to identifying functional lox site domains for the Cre recombinase Sheren, Jamie Langer, Stephen J. Leinwand, Leslie A. Nucleic Acids Res Nucleic Acid Enzymes The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system. Oxford University Press 2007-08 2007-08-15 /pmc/articles/PMC2018622/ /pubmed/17702764 http://dx.doi.org/10.1093/nar/gkm604 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Nucleic Acid Enzymes Sheren, Jamie Langer, Stephen J. Leinwand, Leslie A. A randomized library approach to identifying functional lox site domains for the Cre recombinase |
title | A randomized library approach to identifying functional lox site domains for the Cre recombinase |
title_full | A randomized library approach to identifying functional lox site domains for the Cre recombinase |
title_fullStr | A randomized library approach to identifying functional lox site domains for the Cre recombinase |
title_full_unstemmed | A randomized library approach to identifying functional lox site domains for the Cre recombinase |
title_short | A randomized library approach to identifying functional lox site domains for the Cre recombinase |
title_sort | randomized library approach to identifying functional lox site domains for the cre recombinase |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018622/ https://www.ncbi.nlm.nih.gov/pubmed/17702764 http://dx.doi.org/10.1093/nar/gkm604 |
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