Journal
GENESIS
Volume 39, Issue 4, Pages 256-262Publisher
WILEY
DOI: 10.1002/gene.20053
Keywords
recombinase-mediated cassette exchange; Cre-IoxP; Flp-FRT; ES cells
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Funding
- NCI NIH HHS [CA68485] Funding Source: Medline
- NIDDK NIH HHS [DK20593, DK42502] Funding Source: Medline
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Recombinase-mediated cassette exchange (RMCE), when applied to mouse embryonic stem (ES) cells, promises to increase the ease with which genetic alterations can be introduced into targeted genomic loci in the mouse. However, existing selection strategies for identifying ES cells in which replacement DNA cassettes from a carrier plasmid have been exchanged correctly into a defined locus are suboptimal. Here, we report the generation in mouse ES cells of a loxed cassette acceptor (LCA) allele within the glucokinase (gk) gene locus. Using the gk(LCA) as a test allele, we developed a staggered positive-negative selection strategy that facilitates efficient identification of ES cell clones in which a DNA replacement cassette from a carrier plasmid has been exchanged correctly into the gk(LCA) allele. This selection strategy, by facilitating more efficient production of ES cell clones with various replacement DNA cassettes, should accelerate targeted repetitive introduction of gene modifications into the mouse. (C) 2004 Wiley-Liss, Inc.
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