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 .