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 | Acesso ao texto completo restrito à biblioteca da Embrapa Gado de Leite. Para informações adicionais entre em contato com cnpgl.biblioteca@embrapa.br. |
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Registro Completo |
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Biblioteca(s): |
Embrapa Gado de Leite. |
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Data corrente: |
26/12/2018 |
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Data da última atualização: |
24/01/2023 |
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Tipo da produção científica: |
Artigo em Periódico Indexado |
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Autoria: |
SOUZA, G. T. de; HELL, R. C. R.; SOUZA, J. F. da S.; CAMARGO, L. S. de A. |
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Afiliação: |
LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL. |
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Título: |
Easy in vitro synthesis of optimised functioning reporter mRNA from common eGFP plasmid. |
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Ano de publicação: |
2018 |
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Fonte/Imprenta: |
Molecular Biotechnology, v. 60, n. 10, p. 762-771, 2018. |
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DOI: |
10.1007/s12033-018-0112-5 |
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Idioma: |
Inglês |
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Conteúdo: |
Abstract The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p < 0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5' element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection. MenosAbstract The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which ... Mostrar Tudo |
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Palavras-Chave: |
CRISPR/Cas9; EGFP; GFP; Kozak; MRNA; Sequence. |
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Thesagro: |
RNA. |
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Categoria do assunto: |
X Pesquisa, Tecnologia e Engenharia |
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Marc: |
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024 7 $a10.1007/s12033-018-0112-5$2DOI
100 1 $aSOUZA, G. T. de
245 $aEasy in vitro synthesis of optimised functioning reporter mRNA from common eGFP plasmid.$h[electronic resource]
260 $c2018
520 $aAbstract The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p < 0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5' element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection.
650 $aRNA
653 $aCRISPR/Cas9
653 $aEGFP
653 $aGFP
653 $aKozak
653 $aMRNA
653 $aSequence
700 1 $aHELL, R. C. R.
700 1 $aSOUZA, J. F. da S.
700 1 $aCAMARGO, L. S. de A.
773 $tMolecular Biotechnology$gv. 60, n. 10, p. 762-771, 2018.
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Registro original: |
Embrapa Gado de Leite (CNPGL) |
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| Registros recuperados : 12 | |
| 3. |  | SILVA, A. C. da; CRUZ, E. D.; SOUZA, G. T. de; ALBUQUERQUE, G. D. P. Germinação de sementes de matrizes de taxi-branco Sclerolobium paniculatum Vogel. In: SEMINÁRIO DE INICIAÇÃO CIENTÍFICA, 14., 2010, Belém, PA. Bolsista de iniciação científica: um aporte ao desenvolvimento da pesquisa agropecuária: anais. Belém, PA: Embrapa Amazônia Oriental, 2010. 1 CD-ROM.| Tipo: Artigo em Anais de Congresso |
| Biblioteca(s): Embrapa Amazônia Oriental. |
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| 5. | | MACIEL, P. O.; IWASHITA, M. K. P.; LOPES, L. P. C.; SOUZA, G. T. R. e; TAKEMOTO, R. M. Registro de metacercárias de Austrodiplostomum compactum em olhos de tambacus (Piaractus mesopotamicus x Colossoma macropomum) cultivados no Estado do Tocantins. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE AQUICULTURA E BIOLOGIA AQUÁTICA, 5., 2012, Palmas. Unir, consolidar e avançar: anais. Palmas: AQUABIO, 2012. 1 CD-ROM. Organizado por: Sílvio Ricardo Maurano; AQUACIÊNCIA 2012.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Pesca e Aquicultura. |
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| 7. | | QUINTAO, C. C. R.; ALMEIDA, C. G. de; SOUZA, G. T.; LADEIRA, L. O.; BRANDAO, H. de M.; CAMARGO, L. S. de A.; MUNK, M. Complexação da proteína CAS9 e RNA-GUIA com nanotubos de carbono visando edição gênica em células e embriões de mamíferos. In: SIMPÓSIO NACIONAL DE NANOBIOTECNOLOGIA, 2., 2019, São Bernardo do Campo. Livro de resumos. Santo André: Universidade Federal do ABC, 2018.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Gado de Leite. |
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| 8. | | SOUZA, E. P. D. De; RABELO, N. C.; DIAS, R. B S.; SOUZA, G. T. De; LOURO, I. D.; CAMARGO, L. S. de A. Inibição da HSP90 durante maturação interfere na abundância relativa de transcritos específicos oócito bovino. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE TECNOLOGIA DE EMBRIÕES, 30., 2016, Foz do Iguaçu. Anais... Foz do Iguaçu: Sociedade Brasileira de Tecnologia de Embriões, 2016.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Gado de Leite. |
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| 9. | | LEMOS, D. R.; SOUZA, G. T.; SOUZA, V. G. P.; RIBEIRO, L. S.; SARAIVA, N. Z.; QUINTAO, C. C. R.; CAMARGO, L. S. de A. Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v. 73, n. 1, p. 256-260, 2021. Communication.| Tipo: Nota Técnica/Nota Científica |
| Biblioteca(s): Embrapa Gado de Leite. |
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| 10. | | RIBEIRO, L. dos S.; BRANDÃO, F. Z.; CARVALHEIRA, L. de R.; LEMOS, D. R. de; SOUZA, G. T. de; BATISTA, R. I. T. P.; CAMARGO, L. S. de A.; CARVALHO, B. C. de. Chromium supplementation modulates glucose metabolism in heatstressed Girolando dairy cows. Semina: Ciências Agrárias, v. 41, n. 5, p. 2445-2452, 2020. Short communications.| Tipo: Nota Técnica/Nota Científica |
| Biblioteca(s): Embrapa Gado de Leite. |
|  |
| 11. | | SOUZA, G. T. de; LEMOS, D. R.; SOUZA, V. das G. P. de; SARAIVA, N. Z.; OLIVEIRA, C. S.; QUINTAO, C. C. R.; SIQUEIRA, L. G. B.; CAMARGO, L. S. de A. Pre-implantation development of in vitro fertilized bovine zygotes injected with CRISPR/Cas9 system targeting the betalactoglobulin gene. Animal Reproduction, v. 19, n. 2, e22157, 2022. Edição dos abstracts da Annual Meeting of the Brazilian Embryo Technology Society, 35., Foz do Iguaçu, 2022.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Gado de Leite. |
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| 12. | | SOUZA, V. das G. P. de; SOUZA, G. T. de; LEMOS, D. R. de; GUIMARAES, J. M. de O.; QUINTAO, C. C. R.; MUNK, M.; SARAIVA, N. Z.; CAMARGO, L. S. de A. Heat shock during in vitro maturation of bovine oocytes disturbs bta-miR-19b and DROSHA transcripts abundance after in vitro fertilization. Reproduction in Domestic Animals, v. 56, n. 8, p. 1128-1136, 2021.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 1 |
| Biblioteca(s): Embrapa Gado de Leite. |
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| Registros recuperados : 12 | |
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| Nenhum registro encontrado para a expressão de busca informada. |
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