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Background    Dictyostelium discoideum  amoebae are haploid and, as they share many features with animal cells, should be an ideal creature for studying basic processes such as cell locomotion. Isolation of mutants in this amoeba has largely been limited to non-essential genes:  nsfA —the gene for NEM-sensitive factor—remains the only essential gene for which conditional (ts) mutants exist. These ts mutants were generated by gene replacement using a library of mutagenised  nsfA  containing a selectable marker: transformants were then screened for temperature sensitivity. The success of this approach depended on the high level of homologous recombination prevailing at this locus: ∼95% of selected clones were homologous recombinants. This is unusually high for  Dictyostelium : homologous recombination at other loci is usually much less, usually between 0–30%, making the isolation of ts mutants much more tedious.    Methodology/Principal Findings  In trying to make ts mutants in  sec1A , homologous recombination was found to be only ∼25%. A new approach, involving single  loxP  sites, was investigated.  LoxP  sites are 34 bp sequences recognised by Cre recombinase and between which this enzyme catalyses recombination. A  Dictyostelium  line containing a single  loxP  site adjacent to the 3′ end of the  sec1A  gene was engineered. A  sec1A  replacement DNA also containing a single  loxP  site in a homologous position was then introduced into this cell line. In the presence of CRE recombinase, homologous recombination increased to ∼80% at this locus, presumably largely driven by intermolecular recombination between the two single  loxP  sites.    Conclusions/Significance  A route to increase the rate of homologous recombination at a specific locus,  sec1A , is described which enabled the isolation of 30 ts mutants in  sec1A . One of these,  sec1Ats1 ,has been studied and found to cease moving at the restrictive temperature. The approach described here may be valuable for enhancing homologous recombination at specified loci and thus for introducing mutations into specific genes in  Dictyostelium  and other creatures.

Using Single loxP Sites to Enhance Homologous Recombination: ts Mutants in Sec1 of Dictyostelium discoideum