|
|
Registros recuperados : 310 | |
42. | | RABELO, N. C.; GHETTI, A. M.; CAMARGO, L. S. de A.; VERNEQUE, R. da S.; VIANA, J. H. M. Uso do cultivo overnight na avaliação do potencial de desenvolvimento embrionário e como alternativa na logística da transferência a fresco de embriões F1 Holandês/Gir. In: WORKSHOP DE INICIAÇÃO CIENTÍFICA DA EMBRAPA GADO DE LEITE, 5., 2010, Juiz de Fora, MG. Anais... Juiz de Fora: Embrapa Gado de Leite, 2010. 1 CD-ROM. (Embrapa Gado de Leite. Documentos, 141.). p. 33-36. Biblioteca(s): Embrapa Gado de Leite. |
| |
44. | | JASMIM, J.; MARTINS, S. C.; GONÇALVES, F. S.; ASCARI, I. J.; CAMARGO, L. S. de A.; MENDEZ-OTERO, R. Development of bovine embryons in vitro in co-culture with mesenchymal stem cells and murine embryonic fibroblasts Animal Reproduction, v. 12, n. 3, p. 761. 2015. Edição dos proceedings do 29º Annual Meeting of the Brazilian Embryo Technology Society, 2015, Gramado. Biblioteca(s): Embrapa Gado de Leite. |
| |
47. | | VIANA, J. H. M.; FERREIRA, A. de M.; SÁ, W. F. de; CAMARGO, L. S. de A. Follicular dynamics in zebu cattle. Pesquisa Agropecuária Brasileira, Brasília, DF, v. 35, n. 12, p. 2501-2509, dez. 2000 Título em português: Dinâmica folicular em vacas zebuínas. Biblioteca(s): Embrapa Unidades Centrais. |
| |
48. | | QUINTAO, C. C. R.; OLIVEIRA, C. S.; PEREIRA, M. M.; CAMARGO, L. S. de A.; SARAIVA, N. Z. Effects of forskolin supplementation at different stages of IVP on the preimplantation development of bovine embryos. Animal Reproduction, v. 19, n. 2, e22149, 2022. Edição dos abstracts da Annual Meeting of the Brazilian Embryo Technology Society, 35., Foz do Iguaçu, 2022. Biblioteca(s): Embrapa Gado de Leite. |
| |
50. | | CAMARGO, L. S. de A.; AGUIRRE-LAVIN, T.; ADENOT, P.; ARAUJO, T. D.; SOUZA, E. D.; BEAUJEAN, N. Effect of heat shock during in vitro maturation on heterochromatin compaction in bivone embryos at 4- and 8- cell stages: preliminary study. In: ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, 41., 2015, Versailles. Reproductive performance: at the crossroads of genetics and the environment: abstracts. Versailles: IETS, 2015. p. 132. Biblioteca(s): Embrapa Gado de Leite. |
| |
51. | | ROSA, I. La; CAMARGO, L. S. de A.; PEREIRA, M. M.; FERNANDEZ-MARTIN, R.; PAZ, D. A.; SALAMONE, D. F. Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos. Reproductive Biology and Endocrinology, v. 9, article 18, 2011. Biblioteca(s): Embrapa Gado de Leite. |
| |
53. | | IGUMA, L. T.; FERREIRA, A. de M.; SÁ, W. F. de; VIANA, J. H. M.; CAMARGO, L. S. de A. Manejo reproductivo de rebanõs lecheros. In: MARTINS, P. do C.; DINIZ, F. H.; MOREIRA, M. S. de P.; NOGUEIRA NETTO, V.; ARCURI, P. B. (Ed.). Conocimientos y estratégias tecnológicas para la producción de leche en regiones tropicales. Juiz de Fora: Embrapa Gado de Leite, 2007. p. 289-307. Biblioteca(s): Embrapa Gado de Leite. |
| |
55. | | HIRIART, M. I.; CANEL, N. G.; GAMBINI, A.; BEVACQUA, R. J.; CAMARGO, L. S. de A.; SALAMONE, D. Ooplasmic tranfer on the development of zona-free IVF bovine embryos. Animal Reproduction, v. 10, n. 3, p. 586, Jul./Sept. 2013. Suplemento. Edição dos abstracts do 27º Annual Meeting of the Brazilian Embryo Technology Society, 2013, Praia do Forte, BA. Biblioteca(s): Embrapa Gado de Leite. |
| |
56. | | OLIVEIRA, C. S.; JEDRUSIK, A.; SARAIVA, N. Z.; CAMARGO, L. S. de A.; GARCIA, J. M.; ZERNICKA-GOETZ, M. PAR6C polarity protein expression pattern in male and female bovine embryos. Animal Reproduction, v. 11, n. 3, p. 455, 2014. Edição dos proceedings do 28º Annual Meeting of the Brazilian Embryo Technology Society, 2014, Natal. Biblioteca(s): Embrapa Amazônia Oriental; Embrapa Gado de Leite. |
| |
57. | | VIANA, J. H. M.; PALHÃO, M. P.; SIQUEIRA, L. G. B.; FONSECA, J. F. da; CAMARGO, L. S. de A. Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up. Theriogenology, v. 73, n. 7, p. 966-972, 2010. Biblioteca(s): Embrapa Caprinos e Ovinos; Embrapa Gado de Leite. |
| |
Registros recuperados : 310 | |
|
|
| Acesso ao texto completo restrito à biblioteca da Embrapa Gado de Leite. Para informações adicionais entre em contato com cnpgl.biblioteca@embrapa.br. |
Registro Completo
Biblioteca(s): |
Embrapa Gado de Leite. |
Data corrente: |
26/12/2018 |
Data da última atualização: |
24/01/2023 |
Tipo da produção científica: |
Artigo em Periódico Indexado |
Circulação/Nível: |
B - 1 |
Autoria: |
SOUZA, G. T. de; HELL, R. C. R.; SOUZA, J. F. da S.; CAMARGO, L. S. de A. |
Afiliação: |
LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL. |
Título: |
Easy in vitro synthesis of optimised functioning reporter mRNA from common eGFP plasmid. |
Ano de publicação: |
2018 |
Fonte/Imprenta: |
Molecular Biotechnology, v. 60, n. 10, p. 762-771, 2018. |
DOI: |
10.1007/s12033-018-0112-5 |
Idioma: |
Inglês |
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 |
Palavras-Chave: |
CRISPR/Cas9; EGFP; GFP; Kozak; MRNA; Sequence. |
Thesagro: |
RNA. |
Categoria do assunto: |
X Pesquisa, Tecnologia e Engenharia |
Marc: |
LEADER 03181naa a2200253 a 4500 001 2102521 005 2023-01-24 008 2018 bl uuuu u00u1 u #d 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.
Download
Esconder MarcMostrar Marc Completo |
Registro original: |
Embrapa Gado de Leite (CNPGL) |
|
Biblioteca |
ID |
Origem |
Tipo/Formato |
Classificação |
Cutter |
Registro |
Volume |
Status |
Fechar
|
Nenhum registro encontrado para a expressão de busca informada. |
|
|