title: Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Dept of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. w.leonhard@lumc.nl
authors.forename: Wouter N
authors.initials: WN
authors.lastname: Leonhard
authors.forename: Jeroen H
authors.initials: JH
authors.lastname: Roelfsema
authors.forename: Irma S
authors.initials: IS
authors.lastname: Lantinga-van Leeuwen
authors.forename: Martijn H
authors.initials: MH
authors.lastname: Breuning
authors.forename: Dorien J M
authors.initials: DJ
authors.lastname: Peters
mesh_terms: D000818:Animals; D043171:Chromosomal Instability / Q000235:genetics*; D002872:Chromosome Deletion; D019426:Integrases / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C045073:Cre recombinase; D019426:Integrases