Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection.
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 gkLCA 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 gkLCA 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.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Qiaoming
Q
Long
Kathy D
KD
Shelton
Jill
J
Lindner
Julie R
JR
Jones
Mark A
MA
Magnuson
D000483:Alleles; D000818:Animals; D017931:DNA Primers; D004622:Embryo, Mammalian / Q000166:cytology*; D040461:Gene Components; D018390:Gene Targeting / Q000379:methods*; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D045522:Recombinases / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology*
D017931:DNA Primers; D045522:Recombinases; D005941:Glucokinase