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A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA - BMC Microbiol. (2010)

Bottom Line: The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin.This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements.The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto Carlos Chagas, FIOCRUZ, Curitiba, Parana, Brazil.

ABSTRACT

Background: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.

Results: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.

Conclusions: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

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Related in: MedlinePlus

Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (CmR), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the vector reading frame with start and stop codons are shown.
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Figure 6: Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (CmR), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the vector reading frame with start and stop codons are shown.

Mentions: Three cassettes were inserted into the pBluescript(r) II plasmid (Stratagene, San Diego, USA) following the strategy shown in Figure 6. The cassette containing the neomycin resistance gene (NEO - 800 bp) flanked by a T. cruzi ubiquitin intergenic region (TcUIR - 278 bp) and the cassette containing the T. cruzi Dm28c pol I promoter (617 bp) followed by a TcUIR and a hexahistidine tag were synthesized in vitro (GenScript, Piscataway, USA) (Figure 6). The third DNA segment, represented by the RfA cassette (Invitrogen) (1711 bp), was PCR-amplified from pCR-Blunt and was inserted into pBluescript(r) II KS+. Restriction sites were placed in specific positions of the sequence, to insert the various cassettes or remove some segments of DNA, such that new segments could be inserted for the construction of new vectors.


A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

Batista M, Marchini FK, Celedon PA, Fragoso SP, Probst CM, Preti H, Ozaki LS, Buck GA, Goldenberg S, Krieger MA - BMC Microbiol. (2010)

Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (CmR), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the vector reading frame with start and stop codons are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3020659&req=5

Figure 6: Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (CmR), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the vector reading frame with start and stop codons are shown.
Mentions: Three cassettes were inserted into the pBluescript(r) II plasmid (Stratagene, San Diego, USA) following the strategy shown in Figure 6. The cassette containing the neomycin resistance gene (NEO - 800 bp) flanked by a T. cruzi ubiquitin intergenic region (TcUIR - 278 bp) and the cassette containing the T. cruzi Dm28c pol I promoter (617 bp) followed by a TcUIR and a hexahistidine tag were synthesized in vitro (GenScript, Piscataway, USA) (Figure 6). The third DNA segment, represented by the RfA cassette (Invitrogen) (1711 bp), was PCR-amplified from pCR-Blunt and was inserted into pBluescript(r) II KS+. Restriction sites were placed in specific positions of the sequence, to insert the various cassettes or remove some segments of DNA, such that new segments could be inserted for the construction of new vectors.

Bottom Line: The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin.This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements.The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto Carlos Chagas, FIOCRUZ, Curitiba, Parana, Brazil.

ABSTRACT

Background: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.

Results: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.

Conclusions: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

Show MeSH
Related in: MedlinePlus