Abstract
BackgroundDevelopment 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.ResultsHere 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.ConclusionThis 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.
Highlights
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
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
We have performed a systematic knock-out approach to begin to address the function of genes of unknown function present in Dictyostelium and human but absent from S. cerevisiae and S. pombe [5]
Summary
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. Comparative genomics is based on the conservation of the molecular function of genes in different organisms throughout evolution. Orthologous genes can be studied in simple, genetically tractable model systems, as a first step to address their function in higher organisms, including humans and evaluate their possible roles in diseases. The completion of Dictyostelium genome offers the opportunity to study the function of conserved genes present in the social amoeba and other organisms in a systematic way [1]. Many different aspects of its biology including motility, chemotaxis, cytokinesis, cell-differentiation and morphogenesis among others, are more closely related to those in higher organisms than to unicellular models, such as yeasts [2,3,4].
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