Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function.
BACKGROUND
Development of the post-genomic age in Dictyostelium will require the existence of rapid and reliable methods to disrupt genes that would allow the analysis of entire gene families and perhaps the possibility to undertake the complete knock-out analysis of all the protein-coding genes present in Dictyostelium genome.


RESULTS
Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.


CONCLUSIONS
This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.
Torija
Patricia
P
Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M., Calle Arturo Duperier 4, 28029 Madrid, Spain. ptorija@yahoo.es
Robles
Alicia
A
Escalante
Ricardo
R
D000818:Animals; D017124:Conserved Sequence / Q000502:physiology*; D004023:Dictyostelium / Q000235:genetics* / Q000502:physiology; D018390:Gene Targeting; D005821:Genetic Techniques; D005822:Genetic Vectors; D018503:Genome, Protozoan / Q000502:physiology*; D006801:Humans; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid