Once regions flanking the genes of interest are obtained from the att- PCR amplifications, the knockout DNA constructs can be generated within as few as five days (Figure 5). The BP and LR reactions are robust and have very high success rates; typically, at least 90% colonies screened from our BP and LR reactions are positive. Using the MS/GW knockout
constructs, we successfully obtained dhfr-ts +/- and ech +/- parasites in two different T. cruzi strains. In on-going work, we have used MS/GW constructs to successfully produce single as well as selleck screening library double KO lines for more than 10 other genes, ranging JSH-23 in size from 828 to 2730 nucleotides and up to 3 copies (using additional drug resistance markers). Thus the MS/GW approach appears to be amenable to use as part of a higher throughput gene knockout project. Figure 5 Timeline for constructing a KO plasmids using MS/GW strategy. The Multisite Gateway based method consists of three steps: 1) PCR with attB-containing primers to amplify 5′ and 3′ UTR from genomic DNA; 2) BP recombination
of each PCR products with specific donor vectors to generate entry clones containing the UTRs; 3) LR recombination of the two entry clones made in step 2 and a third entry ARS-1620 mw clone containing Neo/Hyg to create the final construct. (Kan, kanamycin-resistance gene; Amp, ampicillin-resistance gene; Ori, Origin of replication). Overall, the results described here identify the Multisite Gateway (MS/GW) -based system as an efficient tool to create knockout construction for deletion of genes in T. cruzi and should help accelerate the functional analysis of a wider array of genes in this important agent of disease. Conclusion This study documents the development of a
Multisite Gateway based method for efficient gene knockout in T. cruzi. Further, we demonstrate Etofibrate that long-primer-based KO constructs with <80 nucleotides of homologous gene sequences are insufficient for consistent homologous recombination in T. cruzi. The increase in efficiency of gene knockout constructs should facilitate increased throughput for the identification of gene function in T. cruzi using reverse genetics. Methods Culture, transfection and cloning of T. cruzi CL and Tulahuen lines of T. cruzi epimastigotes were cultured at 26°C in supplemented liver digest-neutralized tryptose (LDNT) medium as described previously . A total of 1 × 107 early-log epimastigotes were centrifuged at 1,620 g for 15 min and resuspended in 100 μl room temperature Human T Cell Nucleofector™ Solution (Amaxa AG, Cologne, Germany).