- Methodology article
- Open Access
Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function
© Torija et al; licensee BioMed Central Ltd. 2006
- Received: 25 July 2006
- Accepted: 31 August 2006
- Published: 31 August 2006
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.
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.
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.
- Homologous Recombination
- Bacterial Coloni
- Initial Melting
- Homologous Recombination Event
- Dictyostelium Cell
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 now offers the opportunity to study the function of conserved genes present in the social amoeba and other organisms in a systematic way .
Dictyostelium is a primitive eukaryote, living as a single cell organism while bacteria, its source of nutrients, are present in the soil. When bacteria are consumed, starvation triggers a complex response allowing the cells to aggregate by chemotaxis and form a multicellular structure. 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 [2–4]. 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 .
The classical approach of gene inactivation in Dictyostelium is performed by the insertion of a resistant cassette in the target gene by homologous recombination. Obtaining the disruption construct is time consuming since large flanking sequences are necessary to assure a high efficiency of the process. A PCR-based method and an in vitro transposition strategy have been developed facilitating the technique [6, 7]. In the last method, the cloned gene is subjected to random insertion of a transposon containing a blasticidine-resistance cassette. The construct in which the transposon has interrupted the cloned gene is identified, expanded, digested and used for transformation of Dictyostelium cells. Since transposon insertion is random, it is sometimes necessary to screen a large number of clones, mainly when the gene is small. Besides, after transformation, the variable efficiency of homologous recombination in Dictyostelium makes the isolation of the disruptant strain tedious, since many different transformants must be screened to distinguish between random insertion and homologous recombination. Although all these difficulties are not a threat when few knock-outs are being performed, they can be overwhelming when we try to scale up the number of genes to study. We have therefore optimized all the steps from cloning of the gene, construction of the disruption vector, preparation of DNA for transformation and the screening of Dictyostelium transformants. We describe in detail the methods and illustrate its usefulness to disrupt a subset of genes of unknown function highly conserved between Dictyostelium and human.
Optimizing the construction of disruption vectors in Dictyostelium
We have essentially followed the protocol described by  designed to insert a transposable cassette (EZTN:tetr-bsr), containing blasticidin- and tetracycline-resistance cassettes, into a cloned gene. The gene loci were previously amplified by PCR and cloned into pGEM-t vector as described in the methods section. Ideally, insertion events of the transposon must be in the middle of the clone, leaving large flanking regions to allow for an efficient homologous recombination. Besides, it might be important to interrupt the gene as much 5' as possible to disrupt the coded protein near the N-terminus. Since in vitro transposition is random, many different clones must be analyzed to find a correct location of the cassette. Consequently, the selection of the right insertion might be time-consuming, mainly when the targeted gene is small in comparison with the cloned insert. We have therefore designed a simple PCR strategy for a rapid assessment of the insertion point of the cassette after an in vitro transposition reaction. A single PCR reaction will provide information about the point of insertion and the same reaction can be sequenced directly to obtain the precise location of the insertion.
Analysis of gene disruption by PCR
After selection, transformants were plated in association with Klebsiella aerogenes for clonal isolation. As soon as the lysis plaques are visible, cells from the growing zone were picked up and DNA extracted for PCR analysis. As depicted in Fig 2A,B, two close oligonucleotides surrounding the transposon insertion were previously synthethized for each targeted gene (g3, g4) to allow efficient amplification of the genomic region. A typical example of the analysis of Dictyostelium colonies by PCR is shown in figure 2C. Lanes 1,4,5 and 6 show the absence of the lower band corresponding to the amplification of the unaffected gene with oligos g3 and g4. Concomitantly, an upper band indicates the insertion of the transposon in the locus. An internal control corresponding to the amplification of an unrelated locus is included. The selected strains were grown and saved frozen for future analysis.
Targeting genes of unknown function
Conserved genes of unkown function in Dictyostelium
Dicty gene (Dictybase ID)
Human gene (Genebank #)
Disruption vector (total aa/point of insertion)
A complete protocol from gene cloning to isolation of disruptant strains in Dictyostelium is presented in this methodological report. Every step was optimized from gene cloning to selection of homologous recombination, with the aim to allow large-scale gene disruption strategies, such as the one described for the analysis of genes of unknown function. Once optimized, we have been able to complete the described protocol for a group of 10 genes in approximately 5 weeks of one person's work. It is conceivable for a well-trained technician to process 100 genes in a year. Extrapolating our results, we might expect to fail obtaining knock-out strains in around 30% of any group of genes, in part due to lethality. Other simple approaches must be designed to cope with these difficulties. Meanwhile, the existing methods, such as the one described here, will help to the rapid development of functional genomics in Dictyostelium.
Dictyostelium cell culture
Cells were grown axenically in HL5 medium (14 g/l tryptone, 7 g/l yeast extract, 0.35 g/l Na2HPO4, 1.2 g/l KH2PO4, 14 g/l glucose, pH 6.5) or in association with Klebsiella aerogenes in SM plates (10 g/l glucose, 1 g/l yeast extract, 10 g/l peptone, 1 g/l MgSO4·6H20, 1.9 g/l KH2PO4, 0.6 g/l K2HPO4, 20 g/l agar, pH 6.5) as described by .
PCR-amplification and cloning of the genes of interest
Specific oligonucleotides were designed to amplify a genomic region of 2–3 Kb corresponding to the genes of interest. 1–2 μg of genomic DNA from AX4 was used as template in a standard 50 μl PCR reaction containing dNTPs at 0.5 mM each, oligonucleotides at 1 pmol/μl each, 1.5 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. Different cycle programs were used and the best results were obtained in the following ranges: initial melting: 5 min at 95°C; melting: 1 min at 95°C, annealing: 1 min at 45–50°C, elongation: 5–6 min at 62–65°C, 30 cycles; final elongation: 10 min at 62–65°C. The low extending temperature (62–65°C) allowed the amplification of highly A+T-rich templates as described previously . 5 μl were used for checking the size and purity of the PCR product by agarose gel, the remaining of the reaction was purified with QIAquick PCR purification Kit from Qiagen and eluted in 30 μl. 3 μl were used for cloning into pGEM-T easy vector following manufacture's instructions. Ligation reactions were transformed into E. coli DH5α and the plasmids containing the inserts were recognized by restriction with NotI, which releases the inserted fragment. Plasmid DNA from alkaline mini-preps were purified with QIAquick PCR purification kit from Qiagen and eluted in 30 μl as described by manufacture's instructions. This purification step is essential for the efficiency of the in vitro transposition reaction described below, avoiding the necessity to perform maxi-preparation of the DNA.
Insertion of the transposable cassette into the target gene
10 μg of the EZTN plasmid described by  were digested with PvuII and the reaction was directly purified with QIAquick PCR purification kit from Qiagen and eluted in 30 μl. We found that gel purification of the transposon was not necessary and increased the yield of the procedure. We used 1.5 μl of the pGEMT-cloned insert and 1 μl of the digested transposon in a reaction with 1 μl of transposase from Epicentre in a 5 μl final volume, following the manufacturer's instructions. After performing the transposition reaction and precipitation as described , E. coli DH5α were transformed and plated in LB-agar containing 15 μg/ml tetracycline, 50 μg/ml ampicillin. After 36 hours of incubation at 37°C, 10–20 bacterial colonies from each transposition were analyzed by PCR as described in figure 1. Bacterial colonies were picked up with a pipette tip, resuspended in 20 μl of distilled water and boiled for 5 minutes. 5 μl were used in a 50 μl PCR reaction with universal primers A and B, from pGEMt and EZTN-R oligo (5'-GCCAATATGCGAGAACACCCGAG-3'), derived from the sequence of the transposon . The conditions for the PCR were as follows: 50 μl PCR reaction containing dNTPs at 0.25 mM each, oligonucleotides A, B and EZTN-R at 0.5 pmol/μl each, 1.5 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. The cycling program was as follows: initial melting: 5 min at 95°C; melting, 1 min at 95°C, annealing: 1 min at 50°C, elongation: 2 min at 65°C, 30 cycles; final elongation: 10 min at 65°C. 5 μl of the PCR reactions were used for analysis in agarose gels. The specific insertion point was obtained by direct sequencing of the PCR product with oligo EZTN-R. For this purpose, the remaining 45 μl of the PCR reactions were purified with QIAquick PCR purification kit from Qiagen and eluted in 30 μl. 3 μl were used for sequencing in an Applied Biosystems 377 sequencer. The expected sequence from oligo EZTN-R contains the transposase recognition sequence (which is indicated in Fig 1C), followed by the specific sequence of the targeted gene.
PCR amplification of the whole construct and electroporation of Dictyostelium cells
Selected bacterial colonies containing the plasmid with the inserted transposon were grown overnight in LB supplemented with ampicilin and tetracycline. Alkaline mini-preps were performed and the plasmids digested with NotI for further confirmation. DNA from the mini-preps was purified with QIAquick PCR purification kit. We found this step essential for the quality of the DNA, necessary for PCR amplification in the subsequent steps. The region containing the flanking sequences and the inserted transposon was amplified by PCR using the universal oligonucleotides A and B (see figure 2A,B for a schematic representation). Four PCR reactions of 50 μl were set for each sample as follows: 1 μl of DNA from the purified mini-prep diluted to 1/50; dNTPs at 0.25 mM each, oligonucleotides A and B at 0.5 pmol/μl each, 2 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. Cycling program: initial melting: 5 min at 95°C, melting: 1 min at 95°C, annealing: 1 min at 55°C, elongation: 10 min at 62–65°C, 34 cycles; final elongation: 10 min at 62–65°C. The amplification product is expected to have 5.5–6 Kb (cloned insert plus transposon) and was checked by agarose electrophoresis. The four reactions were joined and purified with QIAquick PCR purification kit. DNA was eluted in 30 μl and 10 μl were electroporated in Dictyostelium cells as described by .
Rapid analysis of gene disruption by PCR
After selection, transformants were plated in association with Klebsiella aerogenes for clonal isolation. As soon as the lysis plaques are visible, cells from the growing zone were picked up with a pipette tip and resuspended in 10 μl of MasterAmp DNA extraction solution from Epicentre. Samples were incubated 10 min at 60°C followed by other incubation of 10 min at 95°C. The DNA prepared in this way will be suitable for PCR amplification to screen for those transformants in which the gene has been disrupted by homologous recombination. 1 μl of the DNA was used in 50 μl PCR reaction containing 0.25 mM dNTPs, oligonucleotides g3, g4 and control oligos for amplification of an unrelated locus, at 0.5 pmol/μl each, 1.25 units of Taq-polimerase (Biotools) and 1× PCR buffer from Biotools. Cycling program: initial melting, 5 min at 95°C; melting: 1 min at 95°C, annealing: 1 min at 45°C, elongation: 3 min at 62°C, 30 cycles; final elongation: 10 min at 62°C. If necessary, this analysis can be performed in pools of clones to allow the rapid assessment of thousands of independent transformants as previously described .
This work was supported by grant BMC2003-02814 from the Spanish Ministerio de Educación y Ciencia. Sequence data for Dictyostelium were obtained from the Genome Sequencing Centers of the University of Cologne, Germany, the Institute of Molecular Biotechnology, Department of Genome Analysis, Jena, Germany, the Baylor Collage of Medicine in Houston, Texas, and the Sanger Center in Hinxton, Cambridge, UK. We thank Carlos Quijano for bioinformatics analysis, Dicty-base team for performing the curated gene models, and Juan J. Vicente and Rosa M. Calvo for critical reading of the manuscript.
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