A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it>
<p>Abstract</p> <p>Background</p> <p>The three trypanosomatids pathogenic to men, <it>Trypanosoma cruzi</it>, <it>Trypanosoma brucei </it>and <it>Leishmania major</it>, are etiological agents of Chagas disease, African sleeping sickne...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2010-10-01
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| Series: | BMC Microbiology |
| Online Access: | http://www.biomedcentral.com/1471-2180/10/259 |
| _version_ | 1818382508177227776 |
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| author | Ozaki Luiz S Preti Henrique Probst Christian M Fragoso Stenio P Celedon Paola AF Marchini Fabricio K Batista Michel Buck Gregory A Goldenberg Samuel Krieger Marco A |
| author_facet | Ozaki Luiz S Preti Henrique Probst Christian M Fragoso Stenio P Celedon Paola AF Marchini Fabricio K Batista Michel Buck Gregory A Goldenberg Samuel Krieger Marco A |
| author_sort | Ozaki Luiz S |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The three trypanosomatids pathogenic to men, <it>Trypanosoma cruzi</it>, <it>Trypanosoma brucei </it>and <it>Leishmania major</it>, 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.</p> <p>Results</p> <p>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<sup>® </sup>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 <it>c-myc </it>epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (<it>Tc</it>Rab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.</p> <p>Conclusions</p> <p>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.</p> |
| first_indexed | 2024-12-14T02:51:35Z |
| format | Article |
| id | doaj.art-5b11f9ad175d4bc3a4e6f75cf060d125 |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-12-14T02:51:35Z |
| publishDate | 2010-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-5b11f9ad175d4bc3a4e6f75cf060d1252022-12-21T23:19:44ZengBMCBMC Microbiology1471-21802010-10-0110125910.1186/1471-2180-10-259A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it>Ozaki Luiz SPreti HenriqueProbst Christian MFragoso Stenio PCeledon Paola AFMarchini Fabricio KBatista MichelBuck Gregory AGoldenberg SamuelKrieger Marco A<p>Abstract</p> <p>Background</p> <p>The three trypanosomatids pathogenic to men, <it>Trypanosoma cruzi</it>, <it>Trypanosoma brucei </it>and <it>Leishmania major</it>, 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.</p> <p>Results</p> <p>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<sup>® </sup>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 <it>c-myc </it>epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (<it>Tc</it>Rab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.</p> <p>Conclusions</p> <p>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.</p>http://www.biomedcentral.com/1471-2180/10/259 |
| spellingShingle | Ozaki Luiz S Preti Henrique Probst Christian M Fragoso Stenio P Celedon Paola AF Marchini Fabricio K Batista Michel Buck Gregory A Goldenberg Samuel Krieger Marco A A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it> BMC Microbiology |
| title | A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it> |
| title_full | A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it> |
| title_fullStr | A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it> |
| title_full_unstemmed | A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it> |
| title_short | A high-throughput cloning system for reverse genetics in <it>Trypanosoma cruzi</it> |
| title_sort | high throughput cloning system for reverse genetics in it trypanosoma cruzi it |
| url | http://www.biomedcentral.com/1471-2180/10/259 |
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