Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection. Genesis 2004 Aug;39(4):256-62
Date
08/03/2004Pubmed ID
15286998DOI
10.1002/gene.20053Scopus ID
2-s2.0-4444252009 (requires institutional sign-in at Scopus site) 19 CitationsAbstract
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.
Author List
Long Q, Shelton KD, Lindner J, Jones JR, Magnuson MAAuthor
Test W. User test user title in the Anesthesiology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AllelesAnimals
DNA Primers
Embryo, Mammalian
Gene Components
Gene Targeting
Glucokinase
Mice
Mutagenesis, Insertional
Plasmids
Recombinases
Stem Cells