PMC:1540739 / 21932-23333 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1540739","sourcedb":"PMC","sourceid":"1540739","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1540739","text":"Rationale for a RMCE-ASAP. Using homologous recombination, the gene of interest (GOI, open boxes: exons), is targeted with a construct introducing upstream of coding regions one loxP (blue arrowhead) and downstream, a positive/negative selection cassette (red box) and a second inverted heterologous loxP (purple arrowhead) to create RMCE-ready ES cells. An exchange is performed in these cells by co-transfecting a Cre expression plasmid and a marker-free plasmid with a floxed mutant GOI (green box: mutated exon), to produce a mutant mouse (path A). Importantly RMCE-ASAP incorporates two major improvements over classical RMCE (path B): (i) the positive/negative selection cassette does not prevent germline transmission, so that RMCE-ready mice can be obtained; (ii) the selection cassette does not replace, but rather lies downstream of the GOI. This is a crucial requirement for accelerated phenotyping in somatic cells, as maintaining a functional GOI ensures that the RMCE-ready locus still behaves like a WT locus. Hence, after breeding the RMCE-ready mouse with mice heterozygotes for the GOI, somatic cells with an RMCE-ready locus and a WT or KO allele can be recovered [e.g. RMCE/+ and RMCE/− mouse embryonic fibroblasts (MEFs)]. Such cells, phenotypically similar to +/+ and +/− cells, can then be used for phenotypic analyses of dominant or recessive mutations after a single exchange.","tracks":[]}