Figure 1A shows the expected genomic loci of dhfr-ts and 1f8Neo in dhfr-ts +/-/Neo parasites. As expected no amplification of the 1f8Neo was observed in Tulahuen WT (wild type) parasites as shown by PCR with primers N1-N2 (Figure 1B). PCR using primers in the flanking genes corroborates the correct insertion of 1f8Neo gene in dhfr-ts +/- parasite’s genome. When using N3-R1, N3-R2 and N3-R3 combinations, bands of 1.9, 2.2 and 2.65 kb respectively, were observed, providing further confirmation that the neomycin phosphotransferase gene (Neo) had been inserted in the correct locus (Figure 1C). The insertion
in the dhfr-ts locus was also confirmed by Southern Blot analysis with gDNA from cloned dhfr-ts +/- and WT parasites digested with SalI and probed with dhfr-ts (Figure 1D). When digested with enzymes SalI and probed LY2874455 nmr with dhfr-ts CDS we observe a band of 3.2 kb in wild type parasites while mutants have a 1092 bp insertion corresponding to the 1f8Neo cassette interrupting the dhfr-ts CDS, resulting in an extra 4.4 kb band in the mutants. Figure 1 Disruption of dhfr-ts using a conventional KO construct pBSdh1f8Neo. A) Diagram of the expected genomic
loci of dhfr-ts and 1f8Neo in dhfr-ts +/-/Neo parasites. B) PCR analysis RAD001 with Neo specific primers of WT Tulahuen and both uncloned and click here selected clones of dhfr-ts +/-/Neo parasites. C) PCR analysis with gDNA from selected clones of dhfr-ts +/-/Neo and WT Tulahuen parasites confirming the expected gene disruption of one allele of the dhfr-ts gene by 1f8Neo. D) Southern Blot analysis of WT Tulahuen and two dhfr-ts +/-/Neo clones digested with SalI and probed with dhfr-ts probe. Diagram not to scale. Numbers are sizes (bp) of expected products. dhfr-ts gene is replaced using a MS/GW construct Since we Farnesyltransferase were able to obtain dhfr-ts +/- parasites we concluded that this gene would be a good
candidate to evaluate the one-step-PCR and Multisite Gateway-based systems for gene knockout constructs in T. cruzi. In the MS/GW recombination fragments, the flanking regions of the gene were used as arms for recombination event, in contrast with the method in Figure 1 where the coding sequence of the gene was used for homologous recombination. Drug resistant lines produced by the transfection of Tulahuen strain epimastigotes with a recombination fragment obtained from pDEST/dhfr-ts_1F8Hyg plasmid (Additional file 2: Figure S2) were cloned and analyzed by PCR and Southern Blot. Figure 2A shows the expected genomic loci of dhfr-ts and 1f8Hyg in the genome of dhfr-ts +/-/Hyg parasites; the results of PCR analysis (Figure 2B) confirm the correct insertion of 1f8Hyg replacing one allele of the dhfr-ts gene (Additional file 3). Southern Blot analysis also showed correct insertion of the 1f8Hyg cassette replacing one copy of the dhfr-ts gene in the genome.