RRC ID 31009
Author Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR.
Title A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system.
Journal Nucleic Acids Res.
Abstract Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small ( approximately 34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.
Volume 32(19)
Pages e143
Published 2004-10-26
DOI 10.1093/nar/gnh136
PII 32/19/e143
PMID 15507682
PMC PMC528815
MeSH Animals Dictyostelium / drug effects Dictyostelium / genetics* Drug Resistance Gene Targeting / methods* Genetic Markers Integrases / metabolism* Mutation Nucleosides / pharmacology Recombination, Genetic Viral Proteins / metabolism*
IF 11.561
Times Cited 132
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Cellular slime molds G90002 G90003