ES2583060T3 - Técnicas mejoradas para la transfección de protoplastos - Google Patents
Técnicas mejoradas para la transfección de protoplastos Download PDFInfo
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- ES2583060T3 ES2583060T3 ES14184365.6T ES14184365T ES2583060T3 ES 2583060 T3 ES2583060 T3 ES 2583060T3 ES 14184365 T ES14184365 T ES 14184365T ES 2583060 T3 ES2583060 T3 ES 2583060T3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8266—Abscission; Dehiscence; Senescence
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Procedimiento para la introducción de una o más moléculas de interés en un protoplasto de célula vegetal que comprende las etapas de: - proporcionar el protoplasto de la célula vegetal mediante degradación enzimática y/o eliminación de la pared celular de una célula vegetal; - realizar una primera transfección del protoplasto de la célula vegetal con una composición que es capaz de inducir una rotura del ADN de doble cadena; - realizar una segunda transfección del protoplasto de la célula vegetal con una o más moléculas de interés, en el que dicha una o más moléculas de interés se seleccionan del grupo que consiste en oligonucleótidos u oligonucleótidos mutagénicos; - permitir que se forme la pared celular; en el que la segunda transfección se lleva a cabo después de la primera transfección, en el que el procedimiento comprende además poner en contacto el protoplasto de la célula vegetal con una composición no enzimática que inhibe o evita la formación (de nuevo) de la pared celular - antes o simultáneamente con la primera transfección; o - entre la primera y la segunda transfección, o - antes o simultáneamente con la segunda transfección y el procedimiento comprende además la etapa de eliminar la composición no enzimática que inhibe o evita la formación de la pared celular - antes o simultáneamente con la primera transfección, o - entre la primera y la segunda transfección, o - antes o simultáneamente con la segunda transfección, o - después de la segunda transfección, y antes de que se deje formar la pared celular.
Description
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Aislamiento de protoplastos de tomate
[0087] Se mantienen cultivos de brotes in vitro de la variedad de cultivo M82 de tomate en medio MS20 suplementado con Micro-agar al 0,8% con un fotoperíodo 16/8 h de 2000 lux a 25ºC y 60-70% de humedad relativa. Se corta suavemente un gramo de hojas jóvenes en CPW9M y se transfiere a la solución de enzima (CPW9M que contiene celulosa de Onozuka RS al 2%, macerozima de Onozuka R10 al 0,4%, 2,4-D (2 mg/ml), NAA (2 mg/ml), BAP (2 mg/ml) pH 5,8), e hidroxiurea (2 mM)). Se deja que proceda la digestión durante la noche a 25ºC en la oscuridad. A la mañana siguiente, se agitan suavemente placas de Petri durante una hora para liberar protoplastos. La suspensión de protoplastos se filtra a través de un tamiz de acero inoxidable de malla de 50 µm y los protoplastos se recogieron por centrifugación a temperatura ambiente durante 5 min. a 85xg. El sedimento de protoplastos se resuspende en CPW9M suplementado con hidroxiurea 2 mM y se añaden 3 ml de CPW18S a la parte inferior de cada tubo. Los protoplastos vivos que se acumulan en la interfase entre las dos capas durante la centrifugación (10 minutos, temperatura ambiente, 85xg) se recogen y se evalúa su densidad utilizando un hemocitómetro. Los protoplastos se recogen por centrifugación durante 5 min a 85x g a temperatura ambiente y se resuspenden en medio MaMG suplementado con hidroxiurea 2 mM hasta una densidad final de 106 por ml.
Transfección de protoplastos de tomates
Formación de la huella genética (Ejemplo 1)
[0088] Para cada transfección, se mezclan 250000 protoplastos con 25 µg de ARN de doble cadena contra Ku70 de tomate y 250 µl de solución PEG (40% PEG4000 (Fluka # 81240), Ca(NO3)2 0,1 M, manitol 0,4 M). Se deja que proceda la transfección durante 20 minutos a temperatura ambiente. Se añaden cinco ml de Ca(NO3)2 0,275 M gota a gota y se mezclan a fondo. Se recogen los protoplastos transfectados por centrifugación durante 5 minutos a 85xg a temperatura ambiente y se lavan dos veces en CPW9M. Finalmente, los protoplastos se resuspenden en K8p suplementado con diclobenilo 2 mg/l e hidroxiurea 2 mM hasta una densidad final de 250.000 por ml y se incuban durante la noche a 25ºC en la oscuridad. La mañana siguiente se recogen los protoplastos por centrifugación a 85xg durante 5 minutos a temperatura ambiente, se lavan una vez en CPW9M suplementado con hidroxiurea 2 mM y se aíslan los protoplastos vivos como se ha descrito anteriormente. Los protoplastos vivos se resuspenden en MaMg hasta una densidad final de 106 por ml y se transfectan como se ha descrito anteriormente con 20 µg de construcción de ZFN (Townsend et al. 2009 Nature). Los protoplastos se sumergen a continuación en alginato y se cultivan en medio de cultivo K8p.
Modificación dirigida de genes (Ejemplo 2)
[0089] Para cada transfección, se mezclan 250000 protoplastos con 25 µg de ARN de doble cadena contra Ku70 de tomate, 20 µg de construcción de ZFN (Townsend et al. 2009 Nature) y 250 µl de solución de PEG (40% PEG4000 (Fluka # 81240), Ca(NO3)2 0,1 M, manitol 0,4 M). Se deja que proceda la transfección durante 20 minutos a temperatura ambiente. Se añaden cinco ml de Ca(NO3)2 0,275 M gota a gota y se mezclan a fondo. Se recogen los protoplastos transfectados por centrifugación durante 5 minutos a 85xg a temperatura ambiente y se lavan dos veces en CPW9M. Finalmente, los protoplastos se resuspenden en K8p suplementado con diclobenilo 2 mg/l e hidroxiurea 2 mM hasta una densidad final de 250.000 por ml y se incuban durante la noche a 25ºC en la oscuridad. La mañana siguiente se recogen los protoplastos por centrifugación a 85xg durante 5 minutos a temperatura ambiente, se lavan una vez en CPW9M suplementado con hidroxiurea 2 mM y se aíslan los protoplastos vivos como se ha descrito anteriormente. Los protoplastos vivos se resuspenden en MaMg hasta una densidad final de 106 por ml y se transfectan como se ha descrito anteriormente con 20 µg de construcción donante. Los protoplastos se sumergen a continuación en alginato y se cultivan en medio de cultivo K8p.
Detección de huellas genéticas (Ejemplo 1)
[0090] Después de 3 días de cultivo, se disuelven discos de alginato en citrato de sodio, los protoplastos se recogen por centrifugación y se congelan en nitrógeno líquido para la posterior extracción de ADN usando el kit de DNAeasy (Qiagen). El marco de lectura abierto de ALS longitud completa se amplifica mediante PCR usando la Taq polimerasa de comprobación de lectura, el producto de PCR se clona en el vector de clonación TOPO XL PCR (Invitrogen) y se transforma en células de E. coli competentes One Shot TOP10 (Invitrogen). Las bacterias se siembran en agar LB suplementado con carbenicilina 100 micras g/ml y se incuban durante la noche a 37ºC. La mañana siguiente, se recogen 400 clones individuales y se utilizan para el análisis de curva de fusión de alta resolución en un aparato Light Cycler (Roche) para identificar clones con un desapareamiento en el locus ALS. Los clones positivos se confirman por secuenciación.
Detección de eventos de modificación dirigida de genes (Ejemplo 2)
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Claims (1)
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imagen1 imagen2
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US28847409P | 2009-12-21 | 2009-12-21 | |
| US288474P | 2009-12-21 | ||
| NL2004020 | 2009-12-24 | ||
| NL2004020 | 2009-12-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2583060T3 true ES2583060T3 (es) | 2016-09-16 |
Family
ID=42237054
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES14184365.6T Active ES2583060T3 (es) | 2009-12-21 | 2010-12-20 | Técnicas mejoradas para la transfección de protoplastos |
Country Status (10)
| Country | Link |
|---|---|
| US (2) | US20130023051A1 (es) |
| EP (2) | EP2813572B1 (es) |
| JP (2) | JP5924773B2 (es) |
| CN (2) | CN102791865B (es) |
| AU (2) | AU2010335107B2 (es) |
| CA (2) | CA2951341A1 (es) |
| DK (1) | DK2813572T3 (es) |
| ES (1) | ES2583060T3 (es) |
| IL (1) | IL220472A (es) |
| WO (1) | WO2011078665A1 (es) |
Families Citing this family (36)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9528124B2 (en) | 2013-08-27 | 2016-12-27 | Recombinetics, Inc. | Efficient non-meiotic allele introgression |
| US10920242B2 (en) | 2011-02-25 | 2021-02-16 | Recombinetics, Inc. | Non-meiotic allele introgression |
| PL2768942T3 (pl) | 2011-10-17 | 2020-05-18 | Massachusetts Institute Of Technology | Dostarczanie dokomórkowe |
| CN103374590B (zh) * | 2012-04-23 | 2015-10-28 | 中国农业大学 | 一种peg介导的原生质体瞬时转化方法及其专用试剂 |
| RU2645475C2 (ru) | 2012-04-25 | 2018-02-21 | Регенерон Фармасьютикалз, Инк. | Опосредованное нуклеазой нацеливание с большими нацеливающими векторами |
| AR091482A1 (es) * | 2012-06-21 | 2015-02-04 | Recombinetics Inc | Celulas modificadas geneticamente y metodos par su obtencion |
| CN105025701B (zh) | 2012-12-27 | 2018-09-25 | 凯津公司 | 去除植物中遗传连锁的方法 |
| WO2014127287A1 (en) * | 2013-02-14 | 2014-08-21 | Massachusetts Institute Of Technology | Method for in vivo tergated mutagenesis |
| WO2014172489A2 (en) | 2013-04-16 | 2014-10-23 | Regeneron Pharmaceuticals, Inc. | Targeted modification of rat genome |
| US10124336B2 (en) | 2013-08-16 | 2018-11-13 | Massachusetts Institute Of Technology | Selective delivery of material to cells |
| CN103468633B (zh) * | 2013-09-24 | 2015-02-25 | 薛刚 | 一种提高川贝母细胞同步化的诱导方法 |
| ES2881473T3 (es) | 2013-10-17 | 2021-11-29 | Sangamo Therapeutics Inc | Métodos de suministro y composiciones para la modificación por ingeniería genética del genoma mediada por nucleasas |
| BR112016013400B1 (pt) | 2013-12-11 | 2023-02-14 | Regeneron Pharmaceuticals, Inc. | Método in vitro para modificar um genoma em um lócus genômico de interesse em uma célula pluripotente |
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| EP1734125A1 (en) * | 2005-06-16 | 2006-12-20 | Institut National De La Recherche Agronomique | Homeologous recombination in MSH2 inactivated plants or cells thereof |
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| US8442563B2 (en) * | 2008-12-11 | 2013-05-14 | Avaya Inc. | Automated text-based messaging interaction using natural language understanding technologies |
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| CA2783551A1 (en) | 2011-06-30 |
| CN104651394A (zh) | 2015-05-27 |
| CA2951341A1 (en) | 2011-06-30 |
| IL220472A (en) | 2015-09-24 |
| JP6053865B2 (ja) | 2016-12-27 |
| US20130023051A1 (en) | 2013-01-24 |
| JP2015165812A (ja) | 2015-09-24 |
| CN102791865A (zh) | 2012-11-21 |
| WO2011078665A1 (en) | 2011-06-30 |
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