Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

Abstract

Background: Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems.

Results: While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes.

Conclusion: Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi.

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 with Neo specific primers of WT Tulahuen and both uncloned and 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.

Figure 2

Replacement of dhfr-ts gene with a MS/GW construct pDEST/dhfr-ts_1F8Hyg. A) Schematic of the expected genomic loci of dhfr-ts and 1f8Hyg in dhfr-ts^+/-/Hyg parasites. B) PCR analysis with gDNA from cloned drug resistant parasites and WT Tulahuen parasites confirm the expected gene deletion of one allele of the dhfr-ts gene and correct insertion of 1f8Hyg. Primer H1 plus the R1, R2 or R3 downstream primers, yield the expected products of 1.8, 2.0 and 2.3 kb, respectively and the combination of H5 plus upstream primers F3, F2 and F1 give the predicted bands of 2.1, 2.4 and 2.8 kb for respectively. See additional file 3: Table S5 for nucleotide sequences of primers. C) Genomic DNA Southern blot analysis of a dhfr-ts^+/-/Hyg Tulahuen clone. gDNA digested with BsrGI and hybridized with labeled Hyg CDS probe. Diagram not to scale. Numbers are sizes (bp) of expected products.

Figure 3

Simultaneous replacement of consecutive ech1 and ech2 genes by a MS/GW construct pDEST/ech-Hyg-GAPDH. A) Diagram of ech1, ech2 and Hyg-GAPDH-IR genomic loci in WT and ech^+/-/Hyg parasites. B) PCR genotyping analysis of: no template control (water); ech^+/-/Hyg (ech^+/-) and WT CL (WT). See additional file 3: Table S5 for nucleotide sequences of primers. C) Southern blot analysis of two clones (2 and 4) of ech^+/-/Hyg. Left panel, gDNA digested with BanI and hybridized with Hyg CDS; right panel, gDNA digested with EcoRI and hybridized with labeled ech1 CDS. Diagrams not to scale. Numbers are sizes (bp) of expected products.

Figure 4

Simultaneous replacement of consecutive ech1 and ech2 genes by another MS/GW construct pDEST/ech_Neo-GAPDH. A) Diagram of ech1, ech2 and Neo-GAPDH 3'UTR genomic loci in ech^+/-/Neo parasites. B) PCR genotyping analysis of: no template control (water); ech^+/-/Neo (ech^+/-) and WT CL (WT). See additional file 3: Table S5 for nucleotide sequences of primers. C) Southern blot analysis of WT CL (WT) and ech^+/-/Neo (ech^+/-) digested with EcoRI and hybridized with Neo CDS. Diagram not to scale. Numbers are sizes (bp) of expected products.

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 clone containing Neo/Hyg to create the final construct. (Kan, kanamycin-resistance gene; Amp, ampicillin-resistance gene; Ori, Origin of replication).