Optimizing the construction of disruption vectors in Dictyostelium

We have essentially followed the protocol described by [7] 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.

Oligonucleotides A, B and EZTN-R (whose location is depicted in Figure 1) were used in a PCR reaction with DNA isolated from the bacterial colonies obtained after in vitro transposition as described in detail in the methods section. In those plasmids where the transposon was inserted in the vector, as illustrated in Fig 1A, a band corresponding to the size of the insert is expected (Fig 1C, lanes 1,3,5). However, if the insertion took place in the insert (see Fig 1B), EZTN-R oligo would be located in the right position for amplification with oligo A or B (depending on the orientation of the transposon). In this case, a band smaller than the size of the insert is expected, and its own size will provide an estimate of the position of the transposon (Fig 1C, lanes 2,4,6,7). In the case shown in Figure 1C, lanes 6 and 7 suggested good candidates. The specific insertion point can then be obtained by direct sequencing of the PCR product with oligo EZTN-R. The use of DNA extracted directly from the bacterial colonies and the procedure to obtained quality DNA for sequencing are optimized steps carefully described in the methods section.

Analysis of gene disruption by PCR

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). Transformation of the PCR product is a faster alternative to the isolation of DNA by maxi-prep and subsequent digestion. Moreover, we have observed a higher transformation efficiency with PCR-derived DNA than that obtained with maxi-preps.

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

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 [9].

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 [10]. 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 [7] 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 [7], 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 [11]. 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 [12].

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 [13].