This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.

<ol>
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The authors have nothing to disclose.

Este protocolo pone de relieve la in vitro e in vivo de utilidad Gluc-SERCaMP para supervisar el agotamiento de calcio ER. Aunque la modificación de proteínas para generar SERCaMP parece generalizarse a otras proteínas reportero 12, elegimos Gaussia luciferasa por su robusta (200-1.000 mayor veces) bioluminiscencia en comparación con otras luciferasas 18. Demostramos detectable inducida thapsigargin de liberación Gluc-SERCaMP a través de un rango de dosis de 100 veces de vir...

Las funciones en muchas capacidades celulares incluyendo plegamiento de proteínas, la secreción de proteínas, homeostasis de los lípidos, y de señalización intracelular 1 retículo endoplasmático (ER). Central a la función normal ER es mantener las concentraciones de calcio luminal a ~ 5.000 veces mayores que se encuentran en el citoplasma 2-4. Este proceso intensivo de energía está regulado por la ATPasa de calcio Sarco / retículo endoplásmico (SERCA), una bomba que mueve iones de calcio en la sala de emergencias. Flujo de calcio desde el ER está mediada principalmente por la rianodina (RyR) e inositol trifosfato (IP3R) receptores. Debido a que muchos procesos ER son dependientes de calcio, lo que altera la tienda puede conducir a estrés ER y la muerte celular eventual. ER desregulación de calcio se ha observado en enfermedades como la cardiopatía, la diabetes, el Alzheimer y el Parkinson 5. Debido a la naturaleza progresiva de estas enfermedades, ha sido un reto para delinear la causa-efecto relación entre patogénesis y alteraciones en la tienda de calcio ER. Varias tecnologías han permitido importantes avances en nuestra comprensión de la dinámica del calcio ER, incluyendo colorantes e indicadores de calcio codificados genéticamente (Gecis). Baja afinidad colorantes de calcio, que aumentan en la fluorescencia cuando se une a Ca 2+, se pueden cargar en las células para examinar compartimentos subcelulares con altas concentraciones de calcio 6. Gecis, tales como D1ER y el receptor permite la monitorización de las fluctuaciones de calcio con un control más preciso de la localización subcelular 7-9. Recientemente, otra clase de Gecis llama indicadores de proteínas de orgánulos atrapado en calcio de medición (CEPIA) se han descrito 10. Un tercer enfoque que combina la genética y la química pequeña molécula es-esterasa específica colorante de carga (TED), que utiliza una carboxilesterasa codificado genéticamente (dirigido a la ER) con un colorante de calcio a base de éster 11. Mientras que el AFORenfoques ementioned tienen fortalezas y debilidades inherentes, pueden proporcionar información valiosa sobre la dinámica del calcio ER a través de mediciones agudos de fluorescencia. Ellos son, sin embargo, no es óptimo para los estudios longitudinales a menudo necesarias para investigar la progresión de la enfermedad. Con el objetivo de diseñar un método para controlar la dinámica del calcio durante períodos prolongados de tiempo, hemos identificado y desarrollado una modificación de proteínas para crear las proteínas secretadas calcio ER monitoreo (SERCaMPs) 12. SERCaMP sortea varias limitaciones asociadas con otras metodologías, proporcionando un método mínimamente invasivo para interrogar en varias ocasiones el almacén de calcio ER. Hemos demostrado anteriormente que la ASARTDL péptido carboxi-terminal (alanina-serina-alanina-arginina-treonina-ácido aspártico-leucina) es suficiente para promover la retención de ER; Sin embargo, bajo condiciones que causan disminuciones en calcio ER, la secuencia del péptido ya no es capaz de retener localizatio ERn y la proteína es secretada 13. La base de la tecnología SERCaMP es el apéndice de ASARTDL a la carboxi-terminal de una proteína secretada (por ejemplo, luciferasa Gaussia, o gluc) de modo que la secreción es provocada por el agotamiento de calcio ER, creando así un reportero robusta de ER disregulación de calcio 12. La expresión de Gluc-SERCaMP a través de métodos transgénicos permite fluidos biológicos incluyendo medio de cultivo celular y plasma a analizar los cambios en la actividad gluc como un indicador de la homeostasis del calcio ER. El método tiene aplicaciones para el estudio longitudinal de alteraciones progresivas en la tienda de calcio ER tanto in vitro como in vivo. El siguiente protocolo se escribe como un esquema general para el uso de SERCaMP basado en gluc para estudiar la homeostasis del calcio ER, pero el protocolo puede servir como una guía para SERCaMPs informadores alternativos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.5mL tubes</td>
			<td>Fisher&#160;</td>
			<td>02-682-550</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP-40 solution&#160;</td>
			<td>Pierce</td>
			<td>28324</td>
			<td>for intracellular GLuc assays</td>
		</tr>
		<tr>
			<td>1mL luer-lok syringes</td>
			<td>Fisher</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL filter tips</td>
			<td>Rainin</td>
			<td>RT-L200F</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 surgical sutures</td>
			<td>Fisher</td>
			<td>NC9598192</td>
			<td></td>
		</tr>
		<tr>
			<td>30g needles</td>
			<td>Fisher Scientific</td>
			<td>14-821-13A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microplate sealing sheets</td>
			<td>Thermo</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads</td>
			<td>Fisher</td>
			<td>22-246-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Auto Flow System</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-AF9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal recovery chamber</td>
			<td>Lyon Vet</td>
			<td>ICU-912-004</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Fisher</td>
			<td>NC9386574</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine growth serum</td>
			<td>Thermo</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine, Native</td>
			<td>Regis Technologies</td>
			<td>1-361204-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting needles 3/8 circle sutures</td>
			<td>WPI</td>
			<td>501803</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital ultrasconic cleaner</td>
			<td>Fisher Scientific</td>
			<td>FS60D</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose, GlutaMAX, pyruvate</td>
			<td>Life Technologies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA mass ladder</td>
			<td>Life Technologies</td>
			<td>10496-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase (recombinant protein)</td>
			<td>Nanolight</td>
			<td>321-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase antibody (for WB, ICC, or IHC)</td>
			<td>New England Biolabs</td>
			<td>E8023S</td>
			<td>1:2000 (WB)</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>CellPoint Scientific</td>
			<td>DS-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluc assay plates (96 well, opaque)</td>
			<td>Fisher</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Allmedtech</td>
			<td>63323-276-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butler Schein</td>
			<td>29404</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>995-2949</td>
			<td></td>
		</tr>
		<tr>
			<td>Kwik Stop Styptic powder</td>
			<td>Butler Schein</td>
			<td>5867</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>a452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge 22R Centrifuge</td>
			<td>Bekman Colter</td>
			<td>368831</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin</td>
			<td>Fisher</td>
			<td>19-898-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stereoscope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Gel and PCR Cleanup</td>
			<td>Machery-Nagel</td>
			<td>740609</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet</td>
			<td>Rainin</td>
			<td>L-200XLS+</td>
			<td></td>
		</tr>
		<tr>
			<td>p24 Lenti-X rapid titer kit</td>
			<td>Clontech</td>
			<td>632200</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR film seal</td>
			<td>Fisher</td>
			<td>AB0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>ReFresh Charcoal Filter canister</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades, #10</td>
			<td>Fine Science tools Inc</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats 150-200g</td>
			<td>Charles River</td>
			<td>Rats</td>
			<td>rats ordered at 150-200g.&#160; Surgery 5 days after arrival</td>
		</tr>
		<tr>
			<td>Small animal ear tags</td>
			<td>National Band and Tag co</td>
			<td>1005-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical drapes</td>
			<td>Braintree Scientific</td>
			<td>SP-MPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 2 plate reader</td>
			<td>BioTek</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Sigma</td>
			<td>T9033</td>
			<td>harmful to human health</td>
		</tr>
		<tr>
			<td>Virapower lentiviral packaging mix</td>
			<td>Life Technologies</td>
			<td>K4975-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xfect Transfection reagent</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Valley Vet</td>
			<td>468RX</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp

El retículo endoplasmático (ER) contiene el mayor nivel de calcio intracelular, con concentraciones aproximadamente 5000 veces mayor que los niveles citoplasmáticos. El control estricto sobre el calcio ER es imprescindible para el plegamiento de proteínas, la modificación y la trata. Las perturbaciones a ER de calcio pueden dar lugar a la activación de la respuesta de la proteína desplegada, un mecanismo de respuesta de estrés ER de tres patas, y contribuir a la patogénesis de una variedad de enfermedades. La capacidad de monitorear alteraciones de calcio ER durante el inicio de la enfermedad y la progresión es importante en principio, pero difícil en la práctica. Los métodos actualmente disponibles para el monitoreo de calcio ER, tales como tintes y proteínas fluorescentes dependientes de calcio, han proporcionado información sobre la dinámica del calcio ER en las células, sin embargo estas herramientas no son muy adecuados para estudios in vivo. Nuestro laboratorio ha demostrado que una modificación en el carboxi-terminal de la luciferasa Gaussia confiere la secreción del reportero en respuesta aEl agotamiento de calcio ER. Los métodos para utilizar una luciferasa basado, proteína de monitoreo de calcio ER secretada (SERCaMP) para in vitro e in aplicaciones in vivo se describen en el presente documento. Este vídeo destaca inyecciones hepáticas, la manipulación farmacológica de Gluc-SERCaMP, extracción de sangre y el procesamiento y los parámetros de ensayo para el control longitudinal de calcio ER.

El método Gluc-SERCaMP permite la evaluación de la homeostasis del calcio ER mediante el muestreo de fluidos extracelulares. Varios controles pueden ser incluidas en el diseño experimental para mejorar la interpretación de los resultados. En primer lugar, el uso de un reportero constitutivamente secretada (por ejemplo, gluc sin la ASARTDL C-terminal o &quot;Gluc-No Tag&quot;) se puede emplear para evaluar los efectos de los tratamientos experimentales en la vía secretora (secreción celular global) y la ex...

Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Monitoreo de la homeostasis del calcio del retículo endoplasmático Usando una<em&gt; Gaussia</em&gt; Luciferasa SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

monitoring-endoplasmic-reticulum-calcium-homeostasis-using-gaussia

Research

JoVE Journal

Biology

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

13.0K vistas.  National Institutes of Health. Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Video: Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

El estudio de biogénesis de la membrana con un ensayo de luciferasa basado en genes

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Investigación

Biología

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Monitoreo de células autónomas Ritmos reloj circadiano de expresión génica utilizando luciferase reporteros Bioluminiscencia

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

La visualización de los ARNm retículo endoplásmico localizados en células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualización del retículo endoplasmático subdominios en células cultivadas

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfectar células RAW264.7 con un gen informador de luciferasa

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

De imagen confocal de medición de las fluctuaciones de calcio en el retículo sarcoplásmico de células musculares lisas cultivadas Uso de FRET a base de

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...