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Method Article
Next generation engineering of conditional mouse alleles with loxP and FRT sites by dual RMCE.
Marco Osterwalder
Developmental Genetics. Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland.
Corresponding Author
Javier Lopez-Rios
Developmental Genetics. Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland.
Corresponding Author
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We have developed dual RMCE (dRMCE) as a rapid, cost-saving and easy to use tool that allows re-engineering the vast majority of existing conditional alleles and generation of mice without the need to resort again to homologous recombination or other more complex or time consuming strategies. dRMCE mobilizes the normal loxP and FRT sites present in most conditional alleles and many gene-trap alleles for site-specific targeting of the endogenous locus with a custom designed replacement cassette. dRMCE is well-suited for the easy and rapid modification of endogenous genes by e.g. inserting molecular tags, introduce disease-causing mutations, swap domains and/or insertion of exogenous genes in mouse ES cell lines. To facilitate access to the technology, we have developed a “dRMCE tool-kit” (available from Addgene) that comprises the dual-recombinase expression vector and several backbone plasmids for easy generation of replacement vectors.
The procedures described here focus mostly on the dRMCE technology. However, we also provide information essential for ES cell culture as far as it is relevant for successful replacement of the locus of interest by dRMCE, generation of highly chimeric mice and germ-line transmission of the modified allele.
This protocol complements the information provided by Osterwalder et al. (Ref.1).
Keywords
dRMCE, conditional alleles, mouse embryonic stem cells, ES cells, targeting frequency, homologous recombination, loxP, FRT, Cre, Flp, recombinase-mediated cassette exchange, RMCE, high-throughput genetic engineering
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Subject Areas
Cell biology
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Molecular Biology
Developmental biology
Biotechnology
Genetics
Associated Publications
Dual RMCE for efficient re-engineering of mouse mutant alleles
by Marco Osterwalder, Antonella Galli, Barry Rosen, +3
Nature Methods (10 December, 2010)
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This is a preprint. It has not completed peer review.
Method Article
Next generation engineering of conditional mouse alleles with loxP and FRT sites by dual RMCE.
Marco Osterwalder
Developmental Genetics. Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland.
Corresponding Author
Javier Lopez-Rios
Developmental Genetics. Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland.
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 16 Dec, 2010
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
We have developed dual RMCE (dRMCE) as a rapid, cost-saving and easy to use tool that allows re-engineering the vast majority of existing conditional alleles and generation of mice without the need to resort again to homologous recombination or other more complex or time consuming strategies. dRMCE mobilizes the normal loxP and FRT sites present in most conditional alleles and many gene-trap alleles for site-specific targeting of the endogenous locus with a custom designed replacement cassette. dRMCE is well-suited for the easy and rapid modification of endogenous genes by e.g. inserting molecular tags, introduce disease-causing mutations, swap domains and/or insertion of exogenous genes in mouse ES cell lines. To facilitate access to the technology, we have developed a “dRMCE tool-kit” (available from Addgene) that comprises the dual-recombinase expression vector and several backbone plasmids for easy generation of replacement vectors.
The procedures described here focus mostly on the dRMCE technology. However, we also provide information essential for ES cell culture as far as it is relevant for successful replacement of the locus of interest by dRMCE, generation of highly chimeric mice and germ-line transmission of the modified allele.
This protocol complements the information provided by Osterwalder et al. (Ref.1).
Figure 1
Figure 2
Associated Publications
Dual RMCE for efficient re-engineering of mouse mutant alleles
by Marco Osterwalder, Antonella Galli, Barry Rosen, +3
Nature Methods (10 December, 2010)