Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive‐negative selection
Author(s) -
Long Qiaoming,
Shelton Kathy D.,
Lindner Jill,
Jones Julie R.,
Magnuson Mark A.
Publication year - 2004
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/gene.20053
Subject(s) - biology , genetics , embryonic stem cell , locus (genetics) , plasmid , allele , gene , stem cell , dna
Abstract 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. genesis 39:256–262, 2004. © 2004 Wiley‐Liss, Inc.