Este trabajo ha sido financiado por la NSF subvención IOB # 0615996.

Parte 1: Preparación de medios de cultivo de nematodos (NGM) placas de Petri Nematodos experimentales, como C. elegans y P. pacificus se suelen cultivar en el laboratorio de 6 cm placas de Petri que contenían medio de nematodos Crecimiento (NGM) 1. Los gusanos se mantienen a 20 ° C, pero se pueden incubar en un rango de temperaturas (entre 4 ° C - 25 ° C), dependiendo de las especies de nematodos y el diseño experimental. Para preparar platos con NGM, las técnicas estériles tienen que ser utilizados para prevenir la contaminación por hongos y bacterias. A continuación se presenta el protocolo para preparar platos NGM: <ol><li> Pesar 15 g de agar, 2,4 g de NaCl, 2 g de triptona, y 2,72 g KH 2 PO 4 en un matraz de 2 L cónica, que el volumen hasta 1 litro con agua, autoclave y deje que se enfríe a 60 ° C en un baño de agua. </li><li> Añadir 0,8 ml cada una de 1 M de CaCl 2, el colesterol (5 mg / ml en etanol), y 1 M MgSO 4. Para prevenir la contaminación por hongos y bacterias, un ml de estreptomicina (100 mg / ml) y 1 ml de nistatina (10 mg / ml) se pueden agregar a cada litro de medio. </li><li> Usando una máquina Unispense y mantener condiciones estériles, vierta los medios de comunicación en los platos que desee. Las placas deben ser llenados con cerca de 2 / 3 de lleno. Una placa estándar de 60 mm de diámetro requiere alrededor de 10 ml de los medios de comunicación. No se mueven las placas hasta que el agar esté completamente gelificada, de lo contrario habrá una superficie irregular de agar, por lo que es difícil de enfocar los gusanos en el microscopio estereoscópico. </li><li> Una vez seco, las placas pueden ser sembradas inmediatamente o almacenarse a 4 ° C hasta la siembra de bacterias. </li></ol> Parte 2: Preparación de las bacterias y OP50 siembra NGM placas de Petri P. pacificus se cultiva en el laboratorio por la alimentación de la cepa de E. coli OP50. OP50 tiene un crecimiento limitado en las placas de NGM, formando un césped fino que facilita la visualización de los gusanos 1. Para cultivar una cultura de la OP50, use LB caldo: <ol><li> Añadir 5 g bactotryptone, 2,5 g de extracto de levadura y 2,5 g de NaCl a una botella de 500 ml con tapón de rosca, completar el volumen a 500 ml con agua destilada y autoclave. </li><li> Inocular el caldo con una sola colonia de OP50 de la placa de cultivo en condiciones estériles y se deja crecer la noche a 37 ° C. </li><li> LB-OP50 está listo para ser utilizado y se puede almacenar a 4 ° C durante varios meses si se mantiene la esterilidad. Cada vez que las placas deben ser cabeza de serie, vierta una pequeña cantidad del caldo en un vaso estéril pequeño y esto ayudará a evitar la contaminación de la cultura de valores. </li><li> A las placas de la semilla, prescindir de 100 a 120 l de la OP50 a los 60 mm de diámetro placa Petri NGM y agitar para que se extendiera. </li><li> Incubar las placas sembradas durante la noche a temperatura ambiente para el césped a crecer. Estas placas sembradas se pueden almacenar a 4 ° C durante un máximo de 3 semanas. El almacenamiento en una posición invertida ayuda a prevenir el secado rápido. </li></ol> Parte 3: Hacer una selección de En esta sección se describe cómo hacer una selección para los gusanos. La recogida se utiliza para transferir los gusanos de un lugar a otro. La selección se hace de calibre 30, un 90% de alambre de platino iridio 10%, aunque algunos investigadores prefieren composiciones de metal un poco diferente: <ol><li> Cortar unos 3-4 cm de alambre, el lugar de 0,5 cm de la misma dentro de la punta de una pipeta Pasteur (cuya punta estaba rota a una menor longitud), y luego se junta en el vaso con un mechero Bunsen. La longitud de los cables que sobresalen de la copa es de unos 3-3,5 cm, pero puede variar de acuerdo a las preferencias individuales. </li><li> Aplanar el extremo del alambre que sobresale con un martillo o unos alicates. Luego, doblar hacia arriba la parte aplanada para formar una bola. </li><li> Suavizar los bordes afilados de la selección con un papel de lija para evitar que se dañe el gusano o el agar. </li></ol> Parte 4: gusanos Picking <ol><li> Para recoger los gusanos, esterilizar el alambre de platino en una llama y arrastre la punta plana de la selección a lo largo del césped bacteriano para cubrir la punta con un grueso bacteria pegajosa, teniendo cuidado de no perforar o dañar la superficie del agar. </li><li> Usando el microscopio estereoscópico, muy ligeramente el pincel punta pegajosa contra el gusano gusanos / para ser recogidos hasta el gusano se pega a las bacterias en la selección. </li><li> Una vez que el gusano está en la punta del pico, se transferirá inmediatamente a la nueva placa de toque o deslizando la punta de la selección contra el césped de bacterias de la placa nueva, y el gusano que se arrastran fuera. Se debe tener cuidado de no dañar la superficie del agar en la placa más que los gusanos se arrastran en los agujeros por lo que es difícil de recuperar. Si el gusano se queda en la selección por mucho tiempo, podría secarse. </li></ol> Parte 5: Sistemas de gestión de mutagénesis Sulfonato de metano etílico (EMS) induce un amplio espectro de mutaciones en el genoma en el 2. Mutantes pueden ser identificados por los defectos, como la puesta de huevos, defecto del músculos, la determinación del sexo, la formación de Dauer, el comportamiento y la formación de las gónadas. El protocolo de mutagénesis EMS es similar a la descrita para el C. elegans 1. <ol><li> Preparar 500 ml (o más) de NaOH 1N. </li><li> Tome 4-5, 6 cm de diámetro, las placas de Petri llenas de gusanos, que contiene unos pocos cientos de gusanos en la tercera larva juvenil (J3) y ​​la etapa de lavado de los gusanos con 2-3 ml de agua estéril M9 por placa. </li><li> Recoge todos los gusanos en un recipiente estéril, desechable 15 ml tubo de centrífuga. Centrifugar a 1500 xg durante 5-7 minutos o hasta que los gusanos forma una bolita. </li><li> Aspirar el líquido y el uso de 2 ml de M9 para volver a suspender los gusanos. Añadir 20 l EMS a un tubo que contiene 2 ml de M9 y agitar para que se disuelva. Después agregar esta solución a la EMS 2 ml de la suspensión de gusano y agitar suavemente. La concentración final de EMS será de 47 mm. Atención: EMS es un mutágeno muy fuerte y debe ser manejado en una campana extractora. Todos los materiales (guantes, pipetas, puntas) que entran en contacto con este mutágeno debe ser tratado con NaOH 1N para inactivar la EMS. Use guantes durante el procedimiento de mutagénesis toda la EMS. Disponer los residuos peligrosos de acuerdo a las directrices de su institución. </li><li> Coloque el tubo de centrífuga y cubierta de la tapa con parafilm y colóquelo horizontalmente en la campana extractora de humos y dejar que los gusanos se incuban durante 3,5 horas. El tubo también puede ser colocado en un agitador de baja velocidad. </li><li> A su vez el tubo y la cubierta de la tapa con parafilm, coloque en posición vertical y se incuban hasta que el disipador gusanos. </li><li> Aspirar el sobrenadante y desecharlo en un vaso / frasco que contiene NaOH 1N. Esto inactivar el SME. </li><li> Añadir 5 ml M9 a los gusanos, invertir el tubo 25-30 veces para lavar los gusanos, se centrifuga a 1.500 g durante 5-7 minutos o hasta que los gusanos forma una bolita. Aspirar el sobrenadante y desecharlo en el vaso de NaOH 1N. </li><li> Repita el paso de lavado por lo menos 3 veces. </li><li> Después del último lavado, vuelva a suspender el gusano de pellets en cantidad mínima (~ 500 l) de la M9 y una pipeta a cabo en placas de NGM contiene un césped de bacterias OP50. En este punto ya no es necesario trabajar en la campana química. </li><li> Deje el líquido en seco durante unos minutos y luego recoger y transferencia de aspecto saludable J4 larvas frescas placas sembradas. Deseche la placa original de acuerdo con las directrices de su institución de residuos peligrosos. </li><li> Deje que el auto gusanos mutagenizadas fertilizar. El uso de un microscopio estereoscópico, la pantalla de los fenotipos de la generación F2 de mutantes recesivos de interés. </li></ol> Parte 6: Psoralen mutagénesis En esta sección se describe el procedimiento para la mutagénesis con TMP en conjunto con luz ultravioleta de onda larga (UV). TMP / UV método de mutagénesis es ampliamente utilizado para inducir mutaciones de deleción en el genoma 3. <ol><li> Recoger los gusanos de 4-5, 6 cm de diámetro, las placas de Petri en un tubo de centrífuga de 15 ml, tal como se describe en el procedimiento anterior. </li><li> Agregar 10 ml de M9, invertir 20 veces para lavar los gusanos, y se centrifuga a 1500 xg durante 5 minutos. Eliminar el sobrenadante y repetir el lavado con un total de 3 veces. Aspirar el líquido y vuelva a suspender los gusanos en alrededor de 10 veces su volumen de M9 que contiene 30 mg / ml de TMP. Pipeta de 20 l de TMP (3 mg / ml) en 2 ml de M9. Como EMS, TMP es un potente cancerígeno y se debe manejar la adopción de medidas de seguridad adecuadas. Para evitar el riesgo potencial de la UV ojos la luz adecuada usa debe ser utilizado. Los materiales desechables se deben desechar en bolsas de bioseguridad adecuadas. </li><li> Incubar el tubo en la oscuridad durante 15 minutos a temperatura ambiente en un agitador de baja velocidad (40 UPM). Ajuste el tubo cubierto verticalmente durante 10 minutos para permitir que todos los gusanos del fregadero. </li><li> Eliminar los gusanos de la parte inferior del tubo con una pipeta Pasteur y transferirlos a una placa de NGM cabeza de serie. Descartar el sobrenadante siguiendo las directrices de su institución químicos peligrosos. </li><li> Mantener la placa en la oscuridad hasta que el exceso de TMP se absorbe en el plato. </li><li> El uso de un medidor de intensidad de los rayos UV, establecer la distancia entre la fuente de luz UV de onda larga y la placa de tal manera que la intensidad de la placa es de 500 mW / cm 2 4. Quite la cubierta de aluminio y la tapa y la tapa y exponer la placa que contiene los gusanos TMP tratados con la radiación UV de onda larga de 50 segundos. </li><li> Tapar y dejar a los gusanos en la oscuridad durante 5 horas a temperatura ambiente. </li><li> Recogida y traslado de aspecto saludable J4 larvas frescas placas sembradas. Deseche la placa original siguiendo las directrices de su institución químicos peligrosos. </li><li> El uso de un microscopio estereoscópico, la pantalla de los fenotipos de la generación F2 de la mutación de interés. </li></ol> Parte 7: Resultados de Representante Algunos de los P. fenotipos mutantes pacificus resultantes de la mutagénesis (ya sea por EMS o TMP / UV) se muestran en la Figura 1. Cada mutante tiene un fenotipo característico que se distingue fácilmente de los animales de tipo salvaje. De los gusanosque fueron tratados con el mutágeno, 30 J3/J4 larvas de la generación parental (P0) se colocaron en platos separados y se les permite poner sus huevos, que son la generación F1. De las placas que mostraban plantas de la F1 saludable, un total de 1000 F1 fueron transferidos a platos individuales y la progenie F2 fue proyectado por los fenotipos. Los animales con Cochambrosa 5,6, falta de coordinación 6 y 7 fenotipos transformador se encontraron. <img src="/files/ftp_upload/2293/2293fig1.jpg" alt="Figura 1" /> Figura 1. Pristionchus pacificus tipo salvaje y los animales mutantes. A. B. hermafrodita peso en peso masculino C. dpy mutante D. unc mutante E. tra mutante. Los dos primeros paneles que muestran de tipo salvaje hermafroditas (izquierda) y machos (derecha) para comparar con los diferentes mutantes en los paneles restantes.

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Cell Biol. 48, 31-31 (1995).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1381-1381 (1994).</li><li>Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. , 1-1 (2005).</li><li>Kenning, C., Kipping, I., Sommer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Genesis. 40 (3), 176-176 (2004).</li><li>Sommer, R. J., Carta, L. K., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental and Applied Nematology. 19 (6), 511-511 (1996).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genes &#38; development. 18 (10), 1198-1198 (2004).</li><li>Wei, A., Yuan, A., Fawcett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 30 (20), 110-110 (2002).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Rev Genet. 3 (5), 356-356 (2002).</li></ol>

Mutagénesis es una técnica ampliamente utilizada para los estudios genéticos en varios organismos modelo experimental. Experimentos de mutagénesis se utilizan a menudo para identificar la función de un gen 2. Los procedimientos de mutagénesis descritos aquí pueden ser utilizados no sólo para P. pacificus, sino también para C. elegans y otras especies de nematodos relacionados. Aquí se describen dos métodos, EMS y mutagénesis TMP / UV. EMS es una susta...

an introduction to worm lab: from culturing worms to mutagenesis

Este protocolo describe los procedimientos para mantener los nematodos en el laboratorio y la forma de mutagenizar utilizando dos métodos alternativos: etil metano sulfonato (EMS) y 4 &quot;, 5, 8 trimetilpsoraleno-combinada con la luz ultravioleta (TMP / UV). Los nematodos son potentes sistemas biológicos para estudios de genética, debido a su plan de cuerpo simple y el sistema de apareamiento, que se compone de auto-fertilización de los hermafroditas y los hombres que pueden generar cientos de progenie por animal. Los nematodos son mantenidos en placas de agar que contiene un césped de bacterias y se pueden transferir fácilmente a partir de un plato a otro con un pico. EMS es un agente alquilante comúnmente utilizadas para inducir mutaciones puntuales y deleciones pequeñas, mientras que TMP / UV induce principalmente supresiones. Dependiendo de las especies de nematodos que se utiliza, las concentraciones de EMS y TMP tendrá que ser optimizada. Para aislar las mutaciones recesivas del nematodo Pristionchus pacificus, los animales de la generación F2 fueron examinados visualmente para fenotipos. Para ilustrar estos métodos, mutagénesis gusanos y buscó falta de coordinación (UNC), Cochambrosa (dpy) y transformador (TRA) mutantes.

Watch this Scientific Journal Video about An Introduction to Worm Lab: from Culturing Worms to Mutagenesis at JoVE.com

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

La detección de mutantes con defectos fenotípicos es un método sencillo para identificar los genes que funcionan en un determinado proceso biológico. En este artículo se describe cómo los gusanos que viven la cultura libre (por ejemplo,<em&gt; Pristionchus pacificus</em&gt;) En el laboratorio y mostrar dos métodos diferentes de mutagénesis, EMS y TMP / UV.

Introducción al Laboratorio de Worm: El cultivo de gusanos de Mutagénesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

an-introduction-to-worm-lab-from-culturing-worms-to-mutagenesis

Research

JoVE Journal

Biology

34.1K vistas.  University of Texas at Arlington. La detección de mutantes con defectos fenotípicos es un método sencillo para identificar los genes que funcionan en un determinado proceso biológico. En este artículo se describe cómo los gusanos que viven la cultura libre (por ejemplo, Pristionchus pacificus) En el laboratorio y mostrar dos métodos diferentes de mutagénesis, EMS y TMP / UV.

Video: An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagénesis dirigida casera de plásmidos enteros

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Investigación

Biología

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Único ovocito Mutagénesis bisulfito

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Introducción a Solid membrana compatibles Basada Electrofisiología

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

En Reprogramación vivo de células somáticas de adultos a Pluripotencia por la sobreexpresión de factores de Yamanaka

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Un In vivo Enfoque de reticulación para aislar Complejos de Proteína Desde Drosophila Los embriones

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

El cultivo<em&gt; Caenorhabditis elegans</em &gt; En axénico Liquid Media y Creación de transgénicos Worms por bombardeo de micropartículas

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

El uso de células tumorales mamarias de ratón para enseñar conceptos básicos de biología: Un módulo de laboratorio simple

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medición de actividad enzimática de la lactasa en el laboratorio de enseñanza

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...