Next generation engineering of conditional mouse alleles with loxP and FRT sites by dual RMCE.
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
Posted 16 Dec, 2010
Next generation engineering of conditional mouse alleles with loxP and FRT sites by dual RMCE.
Posted 16 Dec, 2010
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
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