Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice.
BMC Biotechnol
; 8: 18, 2008 Feb 25.
Article
in En
| MEDLINE
| ID: mdl-18298805
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Recombinant Proteins
/
Mice, Transgenic
/
Protein Engineering
/
Chromosome Deletion
/
Integrases
/
Chromosomal Instability
Limits:
Animals
Language:
En
Journal:
BMC Biotechnol
Journal subject:
BIOTECNOLOGIA
Year:
2008
Document type:
Article
Affiliation country:
Netherlands