LV es apoyado por la NSF (IOS 1002837)

1. Haciendo que el protoplastos <ol><li> Añadir 9 ml de 8% de manitol a una placa de Petri. </li><li> Usando una espátula, coloque siete días de edad musgo 2-3 PPNH4 placas de 5 - en la placa de Petri que contienen manitol. </li><li> Añadir 3 ml de 2% Driselase (Sigma D9515-25G) para la placa de Petri. </li><li> Incubar la placa de Petri a temperatura ambiente con agitación suave durante 1 hora. </li><li> Filtrar la suspensión de protoplastos a través de una malla de 100 micras (BD Falcon 352 350). </li><li> Giro se filtró la suspensión a 250 g durante 5 minutos. Eliminar el sobrenadante. </li><li> Vuelva a suspender los protoplastos con mucha suavidad, con 500 l de 8% de manitol. </li><li> Añadir 9,5 ml de manitol al 8% en el tubo de la cultura. Asegúrese de protoplastos se suspendió totalmente. </li><li> Repita la filtración y la suspensión re-pasos (1.6, 1.7 y 1.8) dos veces más. </li><li> Tomar 10 l de la solución de protoplastos y el recuento de los protoplastos en un hemocitómetro. </li><li> Multiplicar el número de protoplastos en un área de 16 cuadrados por 10.000 para obtener el número de protoplastos por ml (ver Figura 1). </li></ol> 2. Transformación <ol><li> Haga girar la suspensión de protoplastos a 250 g durante 5 minutos. Eliminar el sobrenadante. </li><li> Vuelva a suspender los protoplastos en una solución de MMG en la concentración de 1,6 millones de protoplastos / ml. </li><li> Incubar la suspensión de protoplastos a temperatura ambiente durante 20 minutos. </li><li> Añadir 600 l de suspensión de protoplastos en un tubo de cultivo que contienen 60 mg de ADN. Agitar el tubo suavemente. </li><li> Añadir 700 L de PEG / Ca solución en la mezcla de protoplastos / ADN. Agitar el tubo suavemente hasta que la mezcla es homogénea todo. </li><li> Incubar la mezcla a temperatura ambiente durante 30 minutos. </li><li> Durante este período de espera, la cubierta PRM-B placas con papel de celofán (80 mm de diámetro, los círculos, AA Embalaje Limited, Reino Unido), permitirá celofán para hidratar la superficie de la placa por lo menos durante 5 min. </li><li> Con una espátula, eliminar las burbujas de aire atrapado entre el celofán y la placa. </li><li> Diluir la mezcla con 3 ml de solución W5. </li><li> Haga girar la mezcla a 250 g durante 5 minutos. Eliminar el sobrenadante. </li><li> Vuelva a suspender los protoplastos en derretida 2 ml de PRM-T (antes de añadirla a los protoplastos se puede mantener esta solución líquida a 42 ° C). Placa 1 ml de volver a suspender los protoplastos por PRM-B plato cubierto por papel de celofán. Envuelva las placas con microporos (3M, Cuidado de la Salud). Mantener las placas a 25 ° C en cámara de crecimiento. </li><li> Por otra parte, los protoplastos se resuspendieron en 1 ml de medio de recubrimiento líquido y plateado 0,5 ml en una placa de PRM-B cubiertas con celofán. Esto permite una regeneración más rápida, pero el número de plantas de regeneración es baja. </li><li> Cámara de crecimiento está listo para una hora 16. luz y 8 horas. ciclo de oscuridad. </li><li> Mueva el celofán en una placa nueva selección cuatro días después de la transformación. </li></ol> 3. Los resultados representativos: Un ejemplo de la transformación se muestra en la Figura 2. El ADN utilizado en este ejemplo fue una superenrollado 7,8 kb plásmido que lleva una cinta de resistencia a higromicina. La cantidad de protoplastos plateado se redujo a 50% para la fotografía. Las fotos fueron tomadas dos semanas después de que las plantas fueron trasladadas a las placas de selección. La eficiencia de transformación no se ve afectada por la concentración de ADN de hasta 60 mg. Una mayor concentración de ADN son perjudiciales para la eficiencia de transformación. Los datos mostraron que este método proporciona resultados consistentes en un amplio rango de cantidades de ADN. Este método también puede generar transformantes estables efectivamente como se muestra en la Tabla 1, podemos obtener unos 4 transformantes estables por microgramo de ADN lineal, que es mucho mayor que lo reportado previamente 14. También hemos probado el efecto de choque de calor en la eficiencia de transformación mediante la transformación de un plásmido superenrollado portadores de un gen para una proteína fluorescente como un marcador. El choque térmico se llevó a cabo 10 minutos después de la incubación a temperatura ambiente (en el Paso 2.6) mediante la incubación de la mezcla en un baño de agua a 45 ° C durante 3 minutos. La mezcla se incubó en un baño en la habitación la temperatura del agua inmediatamente después de choque térmico durante 17 min. Los resultados se registraron 22 horas después de la transformación. Las proporciones de los transformantes obtenidos con y sin choque térmico son muy similares (~ 25%), pero se observó un efecto adverso de choque térmico sobre la viabilidad de los protoplastos (datos no mostrados). <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Figura 1" /> Figura 1. Vista de protoplastos en hemocitómetro. La sección necesaria para el recuento de protoplastos se indica mediante un cuadrado rojo. Tenga en cuenta las 16 plazas necesarias para el recuento de protoplastos (ver 1.11), la imagen muestra un recuento de 24 de protoplastos (240.000 células / ml). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Figura 2" /> Figura 2. Fotos de 18 días transitoria transformantes de edad. Protoplastos se transformada con la cantidad indicada de plásmido superenrollado. A: 15μg; B: 30μg; C: 60μg; D: 120μg. <table border="1"><tbody><tr><td> Cantidad de ADN (g) </td><td> La eficiencia (tranformants ADN / mg) </td></tr><tr><td> 15 de plásmido superenrollado </td><td> 120 ± 24 </td></tr><tr><td> 30 de plásmido superenrollado </td><td> 145 ± 54 </td></tr><tr><td> 60, plásmido superenrollado </td><td> 121 ± 60 </td></tr><tr><td> 120, plásmido superenrollado </td><td> 71 ± 38 </td></tr><tr><td> 60, plásmido linealizado </td><td> 4 ± 1 </td></tr></tbody></table> Tabla 1. Transformación de la eficiencia en diferentes cantidades de ADN.

<ol>
	<li>Abel, S., Theologis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Arabidopsis+leaf+protoplasts:+a+versatile+experimental+system+to+study+gene+expression.">Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.</a> Plant J. 5, 421-427 (1994).</li><li>Wood, A. J., Oliver, M. J., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bryophytes+as+model+systems.">Bryophytes as model systems.</a> Bryologist. 103, 128-133 (2000).</li><li>Schaefer, D. G., Zr&#255;d, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+in+the+moss+Physcomitrella+patens.">Efficient gene targeting in the moss Physcomitrella patens.</a> Plant J. 11, 1195-1206 (1997).</li><li>Schaefer, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+in+Physcomitrella+patens.">Gene targeting in Physcomitrella patens.</a> Curr. Opin. Plant Biol. 4, 143-150 (2001).</li><li>Kamisugi, Y., Cuming, A. C., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens.">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Res. 33, e173-e173 (2005).</li><li>Cove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moss+Physcomitrella+patens.">The moss Physcomitrella patens.</a> Annu. Rev. Genet. 39, 339-358 (2005).</li><li>Kamisugi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+gene+targeting+in+Physcomitrella+patens:+homologous+recombination,+concatenation+and+multiple+integration.">The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.</a> Nucleic Acids Res. 34, 6205-6215 (2006).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+formin-mediated+actin-filament+elongation+is+essential+for+polarized+plant+cell+growth.">Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.</a> Proc. Natl. Acad. Sci. U.S.A. , (2009).</li><li>Sawahel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+DNA+into+Physcomitrella+protonemal+tissue+by+using+the+gene+gun.">Transfer of foreign DNA into Physcomitrella protonemal tissue by using the gene gun.</a> Plant Mol. Biol. 10, 315-316 (1992).</li><li>Schaefer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+the+moss+Physcomitrella+patens.">Stable transformation of the moss Physcomitrella patens.</a> Mol. Gen. Genet. 226, 418-424 (1991).</li><li>Cho, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+bombardment+mediated+transformation+and+GFP+expression+in+the+moss+Physcomitrella+patens.">Particle bombardment mediated transformation and GFP expression in the moss Physcomitrella patens.</a> Mol. Cells. 9, 14-19 (1999).</li><li>Ashton, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bryophyte+Physcomitrella+patens+replicates+extrachromosomal+transgenic+elements.">The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements.</a> New Phytol. 146, 391-402 (2000).</li><li>Hohe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+highly+standardized+transformation+procedure+allows+efficient+production+of+single+and+multiple+targeted+gene-knockouts+in+a+moss,+Physcomitrella+patens.">An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.</a> Curr. Genet. 44, 339-347 (2004).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+is+essential+for+tip+growth+in+the+moss+Physcomitrella+patens.">Profilin is essential for tip growth in the moss Physcomitrella patens.</a> Plant Cell. 19, 3705-3722 (2007).</li></ol>

El método aquí presentado permite una transformación de ADN directa y consistente en Physcomitrella patens protoplastos. Este método fue desarrollado originalmente para el 1 de Arabidopsis, pero aquí hemos demostrado que funciona bien con protoplastos de Physcomitrella patens, una planta simple y diferente. Por lo tanto, este método se puede aplicar a otras especies de plantas con modificaciones menores. Posibles modificaciones para la optimización incluyen la concentración de protoplastos en MMG, tiempo de incubación en la solución de MMG y PEG / Ca. Optamos por utilizar 60 mg de ADN en nuestros experimentos de rutina debido a que el número de transformantes se incrementa linealmente con la concentración de ADN hasta este momento (Tabla 1). Podríamos obtener unos 7.000 transformantes transitorios o 200 transformantes estables en una única de 60 mg la transformación del ADN, que permite la detección y análisis de una gran población de plantas. Los protoplastos transformados puede ser extendido con un medio líquido o placas PRM-T. Aunque la eficiencia de la regeneración es más baja cuando el medio de recubrimiento líquido se utiliza, se elige este método cuando los protoplastos se transformaron con plásmidos superenrollado. Como se muestra en la Tabla 1, las transformaciones de hecho con el ADN de plásmido superenrollado generar transformantes muchos más y por lo tanto, podemos obtener un gran número de plantas con el medio de recubrimiento líquido. Además, la difusión de protoplastos con el medio chapado líquido permite una regeneración más rápida, lo cual es importante para los experimentos con fenotipos transitoria, como RNAi tumbar experimentos 9,15. Además de la ausencia de agar para el aislamiento de la parte superior permite planta fácil de inmunotinción y RT-PCR 9,15.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Ingrediente </td></tr><tr><td> PPNH 4 </td><td> 1,84 mM KH 2 PO 4 3,4 mM de Ca (NO 3) 2 1 mM MgSO 4 2,72 mM de tartrato de diamonio 54 M FeSO 4 .7 H 2 O 9,93 M H 3 BO 3 1,97 M MnCl2 0,4 H 2 O 0,23 M CoCl 2 .6 H 2 O 0,19 M ZnSO4. 7H 2 O 0,22 M CuSO 4. 5H 2 O 0,10 M Na 2 MoO 4 .2 H 2 O 0,168 M de KI Agregar 0,7% de agar para un medio sólido. </td></tr><tr><td> PRM-B </td><td> PPNH 4 con 6% de manitol y el 0,8% de agar. Agregar 10 ml de CaCl2 1M a 1 L antes de verter las placas. </td></tr><tr><td> PRM-T </td><td> PPNH 4 con 6% de manitol y el 0,6% de agar. Añadir 0,5 ml de CaCl2 1M a 50 ml antes de su uso. </td></tr><tr><td> Media Revestimiento líquido </td><td> PPNH 4 con el 8,5% de manitol, sin agar. Añadir 0,5 ml de CaCl2 1M a 50 ml antes de su uso. </td></tr><tr><td> 2% Driselase </td><td> Añadir 4 g de Driselase (Sigma D9515-25G) a 200 ml de 8% de manitol. Revuelva suavemente durante 30 minutos. a temperatura ambiente, incubar 30 min. a 4 ° C, y agite suavemente durante 5 minutos. a temperatura ambiente. Girar 2.500 g durante 10 minutos, descartar pellet. Filtro de 0,22 micras, con y almacenar congelados alícuotas de 10 ml. Descongelar en un baño en la habitación la temperatura del agua antes de su uso. </td></tr><tr><td> MMG </td><td> 0,4 M manitol 15 mM MgCl 2 4 mM MES (pH 5.7) </td></tr><tr><td> PEG / Ca </td><td> 4 g PEG4000 3 ml de H 2 O 2,5 ml de 0,8 M de manitol 1 ml de CaCl2 1M </td></tr><tr><td> W5 </td><td> 154 mM NaCl 125 mM de CaCl2 5 mM KCl 2 mM MES (pH 5.7) </td></tr></tbody></table>

efficient polyethylene glycol (peg) mediated transformation of the moss physcomitrella patens

<p class="jove_content"&gt; Un método simple y eficiente para transformar<em&gt; Physcomitrella pantens</em&gt; Protoplastos se describe. Este método es una adaptación de los protocolos para<em&gt; Physocmitrella</em&gt; Protonemal protoplastos y<em&gt; Arabidopsis</em&gt; Mesófilo de transformación de protoplastos<sup&gt; 1</sup&gt;. Debido a su capacidad para someterse a mitótico eficiente recombinación homóloga<em&gt;, Physcomitrella patens</em&gt; Se ha convertido en un importante sistema modelo en los últimos años<sup&gt; 2</sup&gt;. Esta capacidad permite a las altas frecuencias de genes de la orientación<sup&gt; 3.9</sup&gt;, Que no se ve en las plantas de otro modelo, como<em&gt; Arabidopsis</em&gt;. Para sacar el máximo provecho de este sistema, necesitamos un método eficaz y fácil de introducir ADN en las células de musgo. Las formas más comunes para transformar este musgo son bombardeo de partículas<sup&gt; 10</sup&gt; Y PEG-ADN mediada por la captación<sup&gt; 11</sup&gt;. Aunque el bombardeo de partículas puede producir una alta eficiencia de transformación<sup&gt; 12</sup&gt;, Pistolas de genes que no están disponibles para muchos laboratorios y el protocolo es difícil de estandarizar. Por otro lado, la transformación mediada por PEG no requiere de equipos especializados, y se puede realizar en cualquier laboratorio con una campana estéril. Aquí mostramos un método sencillo y altamente eficiente para la transformación de protoplastos de musgo. Este método puede generar más de 120 transformantes transitorios por microgramo de ADN, que es una mejora del protocolo más eficiente ya se ha informado<sup&gt; 13</sup&gt;. Debido a su simplicidad, eficacia y reproducibilidad, este método puede ser aplicado a los proyectos que requieren gran número de transformantes, así como para la transformación de rutina.</p

Watch this Scientific Journal Video about Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens at JoVE.com

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

Un método simple y eficiente para transformar<em&gt; Physcomitrella pantens</em&gt; Protoplastos se describe. Este método es una adaptación de los protocolos para<em&gt; Physocmitrella</em&gt; Protonemal protoplastos y<em&gt; Arabidopsis</em&gt; Mesófilo de transformación de protoplastos<sup&gt; 1</sup&gt;.

Glicol eficiente de polietileno (PEG) de la transformación mediada por Moss Physcomitrella patens</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

efficient-polyethylene-glycol-peg-mediated-transformation-moss

Research

JoVE Journal

Biology

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

40.5K vistas.  Worcester Polytechnic Institute- WPI. Un método simple y eficiente para transformar Physcomitrella pantens Protoplastos se describe. Este método es una adaptación de los protocolos para Physocmitrella Protonemal protoplastos y Arabidopsis Mesófilo de transformación de protoplastos 1.

Video: Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

Inducidas por factores ambientales cambios heredables en lino

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also ...

Investigación

Biología

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

La criopreservación de embriones de ratón por el etileno a base de glicol vitrificación

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Transformación de Genómica de la Picoeukaryote<em&gt; Ostreococcus tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration eficiente de las plantas para la expresión transitoria de alto nivel de proteínas recombinantes

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Eficiente iPS Generación Celular de sangre usando episomas y HDAC inhibidores

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-Transformación de embriones inmaduros mediados de líneas endogámicas de maíz recalcitrante utilizando genes morfogénicos

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Preparación e inyección de embriones de mosquitos Culex para generar mutaciones nulas utilizando CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Agrobacterium-Transformación Genética Mediada, Producción Transgénica, y su Aplicación para el Estudio del Desarrollo Reproductivo Masculino en el Arroz

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

PiggyActivoBac Transformación de la línea germinal mediada por transposasa en el gusano cogollero, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Transformación natural, expresión de proteínas y crioconservación de la filamentosa Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...