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Open Access Methodology article

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice

Wouter N Leonhard1, Jeroen H Roelfsema12, Irma S Lantinga-van Leeuwen1, Martijn H Breuning1 and Dorien JM Peters1*

Author Affiliations

1 Dept of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

2 National Institute of Public Health and the Environment (RIVM), Postbus 1. 3720BA Bilthoven, The Netherlands

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BMC Biotechnology 2008, 8:18  doi:10.1186/1472-6750-8-18

Published: 25 February 2008

Abstract

Background

Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process.

Results

Here, we describe a modification of the multiplex ligation-dependent probe amplification (MLPA), called extension-MLPA (eMLPA), which enables quantification of relatively small differences in DNA that are a consequence of Cre-mediated recombination. eMLPA, here applied on an inducible Pkd1 conditional deletion mouse model, simultaneously measures both the reduction of the floxed allele and the increase of the deletion allele in a single reaction thereby minimizing any type of experimental variation. Interestingly, with this method we were also able to observe the presence of the excised DNA fragment. This extra-chromosomal deletion-circle was detectable up to 5 months after activation of Cre.

Conclusion

eMLPA is a novel strategy which easily can be applied to measure the Cre-mediated recombination efficiency in each experimental case with high accuracy. In addition the fate of the deletion-circle can be followed simultaneously.