Identification of low frequency knockout mutants in Dictyostelium discoideum created by single or double homologous recombination.
Generation and characterization of knockout clones is a widely used approach to evaluate the specific function of a gene product in Dictyostelium discoideum. The mutant clones are generally obtained by double homologous recombination in the target gene. A frequent limitation to obtaining mutants is the low frequency of homologous recombination. Here we present an easy method to identify rare mutants, based on PCR analysis of pools of clones. This method also allows the isolation of functional knockout mutants created by a single homologous recombination event, which can be more frequent than a double recombination event.
Charette
Steve J
SJ
Université de Genève, Centre Médical Universitaire, Département de Physiologie Cellulaire et Métabolisme, Switzerland. charett2@etu.unige.ch
Cornillon
Sophie
S
Cosson
Pierre
P
D000818:Animals; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004023:Dictyostelium / Q000235:genetics*; D005787:Gene Frequency / Q000235:genetics*; D020868:Gene Silencing; D014644:Genetic Variation / Q000235:genetics; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic / Q000235:genetics*