Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
© Xu et al; licensee BioMed Central Ltd. 2009
Received: 18 December 2008
Accepted: 11 May 2009
Published: 11 May 2009
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.
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.
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.
Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease in humans, also known as American trypanosomiasis. T. cruzi infects over 100 species of mammalian hosts and is the leading cause of infection-induced heart failure in Latin America [1, 2]. In 2006, approximately 12,500 deaths have been reported as a result of the clinical complications of T. cruzi-induced heart disease and the lack of effective treatment .
T. cruzi has four morphologically and physiologically distinct stages. The bloodstream trypomastigotes and intracellular amastigotes stages of parasites are in the mammalian host, whereas epimastigotes and metacyclic trypomastigotes develop in the insect vector . The diploid genome of T. cruzi contains approximately 40 chromosomes encoding a predicted set of 22,570 proteins, of which at least 12,570 represent allelic pairs . Allelic copies of genes in the hybrid CL Brener genome may vary in sequence by as much as 1.5%, and trisomy has also been suggested in the case of some chromosomes [6, 7]. Putative functions could be assigned to 50.8% of the predicted protein-coding genes on the basis of significant similarity to previously characterized proteins or known functional domains .
In contrast to the substantial in silico studies of the T. cruzi genome, only 10 genes have been experimentally characterized by reverse genetics in T. cruzi [8–18]. These genes were all disrupted through homologous recombination, using a DNA cassette that has a drug selectable marker flanked by the coding sequence or the untranslated regions (UTRs) of the target gene. Although effective, this conventional gene knockout approach not only requires identification of multiple compatible restriction sites for ligation reactions and for vector linearization, it also involves multiple restriction digestions, ligations and cloning steps that make the process cumbersome and time-consuming . Given that RNA interference has, to date, failed to function in T. cruzi  (in contrast to the situation in the African trypanosomes ), a simplified strategy to knockout genes in T. cruzi would vastly improve the characterization of the multitude of genes encoding proteins without confirmed or even putative functions.
In this study, we sought to develop a simpler method for the deletion of T. cruzi genes. We compared the conventional multi-step knockout technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway (MS/GW) -based systems. We attempted to knockout the dihydrofolate reductase-thymidylate synthase (dhfr-ts) using all three techniques, and enoyl-CoA hydratase (ech) genes using the two alternative approaches. Our results show that gene-specific sequences of 78 nucleotides used in one-step-PCR strategy are not sufficient to guarantee homologous recombination in T. cruzi. However, the MS/GW-based approach is able to efficiently disrupt target genes. In addition, using the MS/GW strategy, generation of knockout constructs can be completed in as few as 5 days. The results of this study will provide a powerful new tool for reverse genetic studies of T. cruzi.
dhfr-ts gene is disrupted using a conventional KO construct
The dhfr-ts gene is annotated as two identical alleles in the diploid CL Brener reference strain and codes for dihydrofolate reductase thymidylate syntase . In most organisms these two enzyme activities are present on separate monofunctional enzymes. In contrast, in T. cruzi both enzymes are on the same polypeptide chain, with the DHFR domain at the amino terminus and the TS domain at the carboxy terminus [22, 23]. Since these enzymes catalyze consecutive reactions in the de novo synthesis of 2'-deoxythymidylate (dTMP), they have been used as targets for chemotherapy, as inhibition of either enzyme disrupts the dTMP cycle and results in thymidine auxotrophy [24–26].
dhfr-ts gene is replaced using a MS/GW construct
Since we 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.
Consecutive ech1 and ech2 genes are simultaneously replaced by constructs generated based on MS/GW system
In T. cruzi, transcript stability and protein translation is largely controlled by 3'UTR and intergenic regions [29, 30]. The intergenic region of a constitutively expressed gene, gapdh, gives consistently high levels of stable RNA in different constructs and in different life cycle stages . Hence, we included the 3' UTR of gapdh in our constructs, to ensure the expression of the inserted drug resistant genes in the epimastigote stage. Transfection of the DNA fragment from pDEST/ech-Hyg-GAPDH (Additional file 4: Figure S3A) resulted in parasite lines that were resistant to Hyg selection. Figure 3A shows the expected genomic loci of ech and Hyg-GAPDH-IR in the genome of ech+/-/Hyg parasites. PCR analysis with the genomic DNA from the drug resistant parasites and WT CL confirmed the expected gene replacement of ech1 and ech2 genes by Hyg-GAPDH-IR (Figure 3B); no products were obtained when using WT CL gDNA as the template with primer combinations f2 and D, f2 and F, C and r2, and E and r2, whereas products of the expected sizes, 1759 bp, 2178 bp, 2696 bp and 2889 bp, respectively, were observed with gDNA from ech+/-/Hyg as the template. Southern blot analysis of EcoR I digested gDNA using the ech1 gene as a probe (Figure 3A and 3C right panel) showed a 4880 bp band corresponding to the replaced allelic copy of both ech genes was undetected in ech+/-/Hyg, whereas the 3490 bp and 1365 bp bands corresponding to the second allele were retained. In addition, a 2988 bp band and a 1478 bp band corresponding to the inserted Hyg-GAPDH-IR were observed in BanI digested gDNA of only the ech+/-/Hyg, but not that of WT CL (Figure 3A and 3C left panel). Taken together, these results confirmed that one copy of each of the tandem ech1 and ech2 genes was replaced by the MS/GW Hyg-GAPDH-IR knockout cassette.
One-step-PCR knockout strategy fails to delete dhfr-ts and ech genes
Since we demonstrated that at least one allele of the dhfr-ts can be deleted using the MS/GW based system, we next tested if this gene can be deleted using the one-step-PCR strategy. Transfection and selection of parasites with the knockout cassette LP-dhfr-ts-Neo failed to yield drug resistant parasites, despite 4 independent attempts. As there are 78 nts of the CDS of dhfr-ts gene in both forward long primers used to produce LP-dhfr-ts-Neo, the drug selectable markers were to be expressed as a fusion protein, with 26 amino acids of the start of dhfr-ts gene fused at the N terminal. It is possible that the knockout parasites were not obtained because the drug selectable marker has reduced enzyme activity when expressed as a fusion protein. To exclude this possibility, we constructed LP-dhfr-ts-UTR-Neo to completely delete the entire dhfr-ts sequence. This construct has 78 nts of the UTR of dhfr-ts gene instead of the CDS, providing production of neomycin phosphotransferase as a non-fusion protein. However, as with the previous construction, no resistant parasites could be obtained despite 4 independent electroporations. Furthermore, one-step-PCR strategy also failed to delete the ech1 and ech2 genes despite 5 independent transfection and selection attempts. Therefore, the constructs generated with one-step-PCR strategy that bear 78 nts gene CDS or UTR specific sequence are likely to be insufficient for homologous recombination in T. cruzi.
Experimental characterization of gene functions in trypanosomatids has relied heavily on reverse genetic approaches and has been facilitated by the development and optimization of gene manipulation strategies and transfection protocols . In contrast to the robust and extensive techniques for genetic manipulation documented in Trypanosoma brucei and Leishmania, the validated techniques and record of success for T. cruzi is much less extensive. A goal of this study was to validate gene KO strategies for T. cruzi which might facilitate research on this important cause of human disease.
Toward that end, we have compared a conventional multi-step cloning technique with two knockout strategies that have been proven to target gene deletion in other organisms, one-step-PCR and MultiSite Gateway. The appeal of the one-step-PCR- strategy is the speed with which constructs can be produced. However, the attempts to knockout either ech or dhfr-ts genes in T. cruzi using this approach were unsuccessful, presumably because the 78 nucleotide-gene-specific regions used in our constructs were insufficient for homologous recombination in T. cruzi. This result is perhaps not surprising as studies in Leishmania  demonstrated that at least 150 nucleotides are needed to guide homologous recombination. However, a recombination rate of 4 × 10-4 was obtained with as short as 42 nucleotides homology in T. brucei . Because of the considerable expense of oligos of >100 bp, we did not investigate the minimum length needed for consistent recombination in T. cruzi, believing such an approach to be impractical for economical, high-efficiency gene knockouts.
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.
This study documents the development of a Multisite Gateway based method for efficient gene knockout in T. cruzi. Further, we demonstrate 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.
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). The resuspended parasites were then mixed with 3–10 μg DNA (8–10 μg DNA for constructs generated using the conventional and MS/GW strategy; 3–10 μg DNA for constructs generated through one-step-PCR) in a total volume of 5–10 μl and electroporated using program "U-33" in an AMAXA Nucleofector Device. This protocol generally yields 6–13% yellow fluorescent protein (YFP) positive parasites 24 hrs after transfection using 10 μg of a YFP-containing control plasmid. The electroporated parasites are cultured in 25 cm2 cell culture flasks (Corning Incorporated, Lowell, MA, USA) with 10 ml LDNT medium; 250 μg/ml G418 (for transfectants with neomycin phosphotransferase gene-containing cassette) and/or 600 μg/ml Hyg (Hygromycin B, for transfectants with hygromycin reisitance gene-containing cassette) was added at 24 hrs post-transfection. Parasites were considered fully selected when parasites transfected with no DNA were dead, generally at 4–5 weeks post-transfection. For single-cell cloning, drug selected lines were deposited into a 96-well plate to a density of 1 cell/well using a MoFlow (Dako-Cytomation, Denmark) cell sorter and cultured in 250 μl LDNT supplemented with G418 or Hyg. Each population from an individual well was considered an individual clone.
Construction of a knockout DNA cassette using the conventional strategy
The complete coding sequence of 1566 bp of the dhfr-ts gene was amplified by PCR from genomic DNA (gDNA) of the WT Tulahuen strain using AmpliTaq Gold® DNA Polymerase (Roche) and primers DH5_f and DH6_r (Additional file 5: Table S1). The PCR product was gel purified with QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA), treated with T4 DNA Polymerase (BioLabs) to generate blunt ends and cloned into the KpnI-digested, T4 DNA Polymerase (BioLabs) treated and dephosphorylated pBlueScript SKII + (Stratagene). Then the dhfr-ts coding region was disrupted by inserting into the PshAI restriction site of the dhfr-ts gene the neomycin phosphotransferase gene which have been generated by digestion with NotI/StuI of pBSSK-neo1f8 plasmid . The resulting recombination vector were sequenced and designated as pBSdh1f8Neo (Additional file 1: Figure S1) containing the Neo CDS plus the trans-splicing 1f8 region, as well as 1016 bp and 550 bp of the 5' and 3'dhfr-ts coding regions.
The final plasmid was digested with restriction enzyme KpnI to liberate the knockout DNA cassette, gel eluted, ethanol precipitated and resuspended in water to a final concentration of 1–2 μg/μl.
Construction of knockout DNA cassettes based on MS/GW strategy
All plasmids were constructed based on MS/GW system using commercially available MultiSite Gateway Three-Fragment Vector Construction kit (Invitrogen, Carlsbad, CA, USA), which includes all the Entry vectors and Destination vectors used in this study (Figure 5). In the Gateway system, the "BP" reaction is a recombination reaction between attB and attP sites in the PCR fragment and Donor vectors, resulting in Entry clones contains the gene of interest flanked by attL sites. "LR" reactions allow recombination between attL and attR sites of a Destination vector to yield an expression clone.
In order to construct the pDEST/dhfr-ts_1F8Hyg plasmid, 1.0-kb 5' flanking sequence of dhfr-ts was amplified from gDNA of the WT CL strain using primers attB4_5'UTR_dhfr_f and attB1_5'UTR_dhfr_r (Additional file 6: Table S2) and Platinum® PCR SuperMix (Invitrogen), gel purified with QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA) and cloned into the Entry vector pDONR™P4-P1R through a BP reaction using the BP Clonase II Enzyme Mix (Invitrogen), resulting in the Entry clone pDONR_5'UTR_dhfr. Similarly, 1.0-kb 3' flanking sequence of dhfr-ts was amplified using primers attB2_3'UTR_dhfr_f and attB3_3'UTR_dhfr_r (Additional file 6: Table S2) and cloned into pDONR™P2R-P3 to generate pDONR_3'UTR_dhfr. Using plasmid pBSSK-hyg1f8  as a template, the Hyg and its upstream 1f8 region was amplified with primers attB1_1F8_f and attB2_1F8Hyg_r (Additional file 6: Table S2) and cloned into Entry vector pDONR™221. The three Entry clones were then mixed with a Destination vector pDEST™R4-R3 in an LR reaction using the LR Clonase II Plus Enzyme Mix (Invitrogen) to generate a final plasmid pDEST/dhfr-ts_1F8Hyg (Additional file 2: Figure S2). The knockout DNA cassette was liberated from the plasmid backbone with AlwNI and PvuI enzymes, and purified as above.
pDEST/ech _Neo-GAPDH and pDEST/ech _Hyg-GAPDH
Trypanosoma cruzi ech1 and ech2 are tandemly arranged genes. To construct the pDEST/ech _Hyg-GAPDH plasmid, 1.0-kb 5' sequence of ech2 was amplified with primers attB4_ech5'UTR_f and attB1_ech5'UTR_r (Additional file 6: Table S2), gel purified and cloned into the Entry clone pDONR-ech5'UTR. Similarly, 1.0-kb 3' sequence of ech1 was amplified with primers attB2_ech3'UTR_f and attB3_ech3'UTR_r (Additional file 6: Table S2) and cloned into pDONR™P2R-P3 to generate pDONR-ech3'UTR. Hyg and the downstream intergenic region of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (GAPDH-IR) was amplified from plasmid pTEX-Hyg.mcs  using primers attB1_Hyg_f and attB2_Hyg_r (Additional file 6: Table S2) and cloned into Entry vector pDONR™221. The three Entry clones were then mixed with a Destination vector pDEST™R4-R3 to generate pDEST/ech _Hyg-GAPDH (Additional file 4: Figure S3A) through a LR reaction. The final plasmid was digested with restriction enzymes PvuII and PciI and purified as above.
Similarly, to construct pDEST/ech _Neo-GAPDH (Additional file 4: Figure S3B), Neo and 3'UTR of GAPDH (GAPDH 3'UTR) was amplified from plasmid pTrex-YFP (modified from the backbone of pTrex ) with primers attB1_Neo_f and attB2_Neo_r (Additional file 6: Table S2) and cloned into Entry vector pDONR™221. The final plasmid was digested with restriction enzymes PvuI and PciI and purified as above.
Construction of knockout DNA cassettes via one-step-PCR
For the constructs for deletion of the dhfr-ts gene using one-step-PCR, Neo and Hyg was amplified with primers LP_dhfr_Neo_f and LP_dhfr_Neo_r, and LP_dhfr_Hyg_f and LP_dhfr_Hyg_r (Additional file 7: Table S3) from plasmids pTrex-YFP and pTEX-Hyg.mcs respectively. In both cases, forward primers and reverse primers corresponded to the 78 bp downstream of the start codon of the dhfr-ts gene and reverse 78 bp upstream of the stop codon of the gene, respectively.
In addition, primers LP_dhfr-UTR_Neo_f and LP_dhfr-UTR_Neo_r, (Additional file 7: Table S3) were also used to amplify Neo from pTrex-YFP. In this case, LP_dhfr-UTR_Neo_f included 78 bp upstream of the start codon of the dhfr-ts gene whereas LP_dhfr-UTR_Neo_r bears 78 bp downstream of the stop codon.
Likewise, primers LP_ech_Neo_f and LP_ech_Neo_r (Additional file 7: Table S3) were designed to amplify the final construction for deletion of the ech genes as well as primers LP_ech_Hyg_f and LP_ech_Hyg_r (Additional file 7: Table S3). PCR reactions were carried out as follows: initial denaturation at 94°C for 3 min followed by 30 cycles of: 98°C for 20s; 55°C for 30s; and 72°C for 2 min followed by 72°C for 10 min using Gradient Master Thermocycler (Eppendorf, Westbury, NY, USA). Products were collected and purified with QIAquick PCR Purification Kit. The eluted DNA was further ethanol precipitated and resuspended to 0.2–1 μg/μl.
For Southern blot analysis, gDNA from different clones and strains was purified using Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). The DNA was digested and separated by 0.7% agarose gel electrophoresis, and the gels blotted onto nylon membranes (Hybond-N 0.45-mm-pore-size filters; Amersham Life Science) using standard methods . For probe generation, a 1030 bp DNA (Hyg) was amplified using primers Hyg_f and Hyg_r (Additional file 8: Table S4) from plasmid pTEX-Hyg.mcs. For the Neo probe, a 795 bp DNA fragment was amplified from plasmid pBSSK-neo1f8 using primers Neo_f and Neo_r (Additional file 8: Table S4). ech1 gene were amplified using primers ech1_pb_f and ech1_pb_r (Additional file 8: Table S4) from gDNA of WT CL, while dhfr-ts gene was amplified from gDNA of WT Tulahuen using primers DH5_f and DH6_r (Additional file 5: Table S1). The PCR products were purified as above. Labeling of the probe and DNA hybridization were performed according to the protocol supplied with the PCR-DIG DNA-labeling and detection kit (Roche Applied Science, Indianapolis, IN, USA).
We are grateful to Dr. Angel M. Padilla and Dr. Todd Minning for valuable comments throughout this study. We would like to thanks Dr. Mirella Ciaccio for her help in the initial steps of the work with the dhfr-ts gene, Dr. Antonio Gonzalez for facilitating the construction of the plasmids pBSSK-neo1f8 and pBSSK-hyg1f8, Dr. Becky Bundy, Courtney Boehlke and Laura Simpson for their technical assistance, and Daniel B. Weatherly for bioinformatics expertise. This work was supported by NIH Grant PO1 AI0449790 to RLT.
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