Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection
New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the tr...
- Autores:
-
Pacheco-Lugo, Lisandro
Díaz-Olmos, Yirys
Sáenz-García, José
- Tipo de recurso:
- Fecha de publicación:
- 2017
- Institución:
- Universidad Simón Bolívar
- Repositorio:
- Repositorio Digital USB
- Idioma:
- eng
- OAI Identifier:
- oai:bonga.unisimon.edu.co:20.500.12442/1877
- Acceso en línea:
- http://hdl.handle.net/20.500.12442/1877
- Palabra clave:
- Transfection
Nucleofection
Electroporation
Trypanosoma cruzi
- Rights
- License
- Licencia de Creative Commons Reconocimiento-NoComercial-CompartirIgual 4.0 Internacional
| id |
USIMONBOL2_b3e849cb81684d7385ff06e5a60d6ce4 |
|---|---|
| oai_identifier_str |
oai:bonga.unisimon.edu.co:20.500.12442/1877 |
| network_acronym_str |
USIMONBOL2 |
| network_name_str |
Repositorio Digital USB |
| repository_id_str |
|
| dc.title.eng.fl_str_mv |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| title |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| spellingShingle |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection Transfection Nucleofection Electroporation Trypanosoma cruzi |
| title_short |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| title_full |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| title_fullStr |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| title_full_unstemmed |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| title_sort |
Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection |
| dc.creator.fl_str_mv |
Pacheco-Lugo, Lisandro Díaz-Olmos, Yirys Sáenz-García, José |
| dc.contributor.author.none.fl_str_mv |
Pacheco-Lugo, Lisandro Díaz-Olmos, Yirys Sáenz-García, José |
| dc.subject.eng.fl_str_mv |
Transfection Nucleofection Electroporation Trypanosoma cruzi |
| topic |
Transfection Nucleofection Electroporation Trypanosoma cruzi |
| description |
New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (N90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce. |
| publishDate |
2017 |
| dc.date.issued.none.fl_str_mv |
2017-01 |
| dc.date.accessioned.none.fl_str_mv |
2018-03-20T20:58:15Z |
| dc.date.available.none.fl_str_mv |
2018-03-20T20:58:15Z |
| dc.type.eng.fl_str_mv |
article |
| dc.type.coar.fl_str_mv |
http://purl.org/coar/resource_type/c_6501 |
| dc.identifier.issn.none.fl_str_mv |
13835769 |
| dc.identifier.uri.none.fl_str_mv |
http://hdl.handle.net/20.500.12442/1877 |
| identifier_str_mv |
13835769 |
| url |
http://hdl.handle.net/20.500.12442/1877 |
| dc.language.iso.spa.fl_str_mv |
eng |
| language |
eng |
| dc.rights.coar.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| dc.rights.license.spa.fl_str_mv |
Licencia de Creative Commons Reconocimiento-NoComercial-CompartirIgual 4.0 Internacional |
| rights_invalid_str_mv |
Licencia de Creative Commons Reconocimiento-NoComercial-CompartirIgual 4.0 Internacional http://purl.org/coar/access_right/c_abf2 |
| dc.publisher.eng.fl_str_mv |
Japanese Society of Parasitology |
| dc.source.eng.fl_str_mv |
Parasitology International |
| dc.source.spa.fl_str_mv |
Vol. 66, No.3 (2017) |
| institution |
Universidad Simón Bolívar |
| dc.source.uri.none.fl_str_mv |
https://www.sciencedirect.com/science/article/pii/S1383576916304330 |
| bitstream.url.fl_str_mv |
https://bonga.unisimon.edu.co/bitstreams/5ec6ee34-bf44-4662-82aa-af5889025293/download |
| bitstream.checksum.fl_str_mv |
8a4605be74aa9ea9d79846c1fba20a33 |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 |
| repository.name.fl_str_mv |
DSpace UniSimon |
| repository.mail.fl_str_mv |
bibliotecas@biteca.com |
| _version_ |
1814076165149687808 |
| spelling |
Licencia de Creative Commons Reconocimiento-NoComercial-CompartirIgual 4.0 Internacionalhttp://purl.org/coar/access_right/c_abf2Pacheco-Lugo, Lisandro4ffb7ad5-96a8-4959-a2e5-59d447d8e253-1Díaz-Olmos, Yirysa494db94-d580-4f7b-ab06-58058add81b7-1Sáenz-García, Joséd76eb640-8f14-4f73-8603-7fa5e4b2e127-12018-03-20T20:58:15Z2018-03-20T20:58:15Z2017-0113835769http://hdl.handle.net/20.500.12442/1877New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (N90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce.engJapanese Society of ParasitologyParasitology InternationalVol. 66, No.3 (2017)https://www.sciencedirect.com/science/article/pii/S1383576916304330TransfectionNucleofectionElectroporationTrypanosoma cruziEffective gene delivery to Trypanosoma cruzi epimastigotes through nucleofectionarticlehttp://purl.org/coar/resource_type/c_6501J.R. Coura, P.A. Viñas, Chagas disease: a new worldwide challenge, Nature 465 (2010) S6–S7, http://dx.doi.org/10.1038/nature09221.N.M. El-Sayed, P.J.Myler, D.C. Bartholomeu, D. Nilsson, G. Aggarwal, A.-N. Tran, et al., The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease, Science 309 (2005) 409–415, http://dx.doi.org/10.1126/science.1112631.H. Ngô, C. Tschudi, K. Gull, E. Ullu, Double-stranded RNA induces mRNA degradation in Trypanosoma brucei, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 14687–14692, http:// www.ncbi.nlm.nih.gov/pubmed/9843950.W.D. DaRocha, K. Otsu, S.M.R. Teixeira, J.E. Donelson, Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi, Mol. Biochem. Parasitol. 133 (2004) 175–186, http://www. ncbi.nlm.nih.gov/pubmed/14698430.C. Subramaniam, P. Veazey, S. Redmond, J. Hayes-Sinclair, E. Chambers, M. Carrington, K. Gull, K. Matthews, D. Horn, M.C. Field, Chromosome-wide analysis of gene function by RNA interference in the African trypanosome, Eukaryot. Cell. 5 (2006) 1539–1549, http://dx.doi.org/10.1128/EC.00141-06.J.C. Morris, Z. Wang, M.E. Drew, P.T. Englund, Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library, EMBO J. 21 (2002) 4429–4438, http://www.ncbi.nlm.nih.gov/pubmed/12198145.S. Alsford, D.J. Turner, S.O. Obado, A. Sanchez-Flores, L. Glover, M. Berriman, C. Hertz- Fowler, D. Horn, High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome, Genome Res. 21 (2011) 915–924, http://dx.doi.org/10.1101/gr.115089.110S. Alsford, S. Eckert, N. Baker, L. Glover, A. Sanchez-Flores, K.F. Leung, D.J. Turner, M.C. Field, M. Berriman, D. Horn, High-throughput decoding of antitrypanosomal drug efficacy and resistance, Nature 482 (2012) 232–236, http://dx.doi.org/10. 1038/nature10771.D. Peng, S.P. Kurup, P.Y. Yao, T.A. Minning, R.L. Tarleton, CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi, MBio 6 (2015) http://dx.doi.org/10.1128/mBio.02097-14 e02097-2014.N. Lander, Z.-H. Li, S. Niyogi, R. Docampo, CRISPR/Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment, MBio 6 (2015) e01012–e01015, http://dx.doi.org/10.1128/mBio. 01012-15.G. de A. Burle-Caldas, V. Grazielle-Silva, L.A. Laibida, W.D. DaRocha, S.M.R. Teixeira, Expanding the tool box for genetic manipulation of Trypanosoma cruzi, Mol. Biochem. Parasitol. 203 (2015) 25–33, http://dx.doi.org/10.1016/j.molbiopara.2015. 10.004.G. Burkard, C.M. Fragoso, I. Roditi, Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei, Mol. Biochem. Parasitol. 153 (2007) 220–223, http://dx.doi.org/10.1016/j.molbiopara.2007.02.008.G. Schumann Burkard, P. Jutzi, I. Roditi, Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters, Mol. Biochem. Parasitol. 175 (2011) 91–94, http://dx.doi.org/10.1016/j.molbiopara.2010.09.002.P.K. Padmanabhan, R.B. Polidoro, N.S. Barteneva, R.T. Gazzinelli, B.A. Burleigh, Transient transfection and expression of foreign and endogenous genes in the intracellular stages of Trypanosoma cruzi, Mol. Biochem. Parasitol. 198 (2014) 100–103, http://dx.doi.org/10.1016/j.molbiopara.2015.02.001.W.D. DaRocha, R.A. Silva, D.C. Bartholomeu, S.F. Pires, J.M. Freitas, A.M.Macedo, M.P. Vazquez, M.J. Levin, S.M.R. Teixeira, Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols, Parasitol. Res. 92 (2004) 113–120, http://dx.doi.org/10.1007/s00436-003-1004-5.M.P. Vazquez, M.J. Levin, Functional analysis of the intergenic regions of TcP2beta gene loci allowed the construction of an improved Trypanosoma cruzi expression vector, Gene 239 (1999) 217–225.D. Xu, C. Pérez Brandán, M. Basombrío, R.L. Tarleton, Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi, BMC Microbiol. 9 (2009) 90, http://dx.doi.org/10.1186/1471-2180-9-90.S. Su, B. Hu, J. Shao, B. Shen, J. Du, Y. Du, J. Zhou, L. Yu, L. Zhang, F. Chen, H. Sha, L. Cheng, F. Meng, Z. Zou, X. Huang, B. Liu, CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients, Sci. Rep. 6 (2016) 20070, http://dx.doi.org/10.1038/srep20070.L. Chicaybam, A.L. Sodre, B.A. Curzio, M.H. Bonamino, An efficient low cost method for gene transfer to T lymphocytes, PLoS One 8 (2013), e60298. http://dx.doi.org/ 10.1371/journal.pone.0060298.LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://bonga.unisimon.edu.co/bitstreams/5ec6ee34-bf44-4662-82aa-af5889025293/download8a4605be74aa9ea9d79846c1fba20a33MD5220.500.12442/1877oai:bonga.unisimon.edu.co:20.500.12442/18772019-04-11 21:51:27.293metadata.onlyhttps://bonga.unisimon.edu.coDSpace UniSimonbibliotecas@biteca.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 |