Whitehead Institute Fellows Funds

Part 1: cDNA synthesis from RNA
<ol>
<li>Before using the Ambion Amino Allyl MessageAmp II kit, add the recommended volume of 100% ethanol to the wash buffers.</li>
<li>In PCR reaction tube, add between 100ng to 5&micro;g of total RNA and 1&micro;l of T7 oligo (dT) primer. Bring the volume up to 12&micro;l with nuclease-free water.</li>
<li>Incubate samples at 70&deg;C for 10 min in a thermocycler.</li>
<li>Remove RNA samples from 70&deg;C and centrifuge briefly. Place on ice.</li>
<li>Prepare the 1st Strand Synthesis master mix and keep at room temperature (with 10% overage for pipetting error). (Table 1)</li>
<li>Gently pipette Master Mix or flick to mix, and then centrifuge briefly.</li>
<li>Transfer 8&micro;l of the Master Mix to each RNA sample. Mix by pipetting up and down 3-4 times.</li>
<li>Incubate at 42&deg;C for 2 hours in thermocycler.</li>
<li>Centrifuge samples briefly and place on ice. Immediately proceed to the next step of dsDNA synthesis.</li>
<li>Prepare the 2nd Strand Synthesis master mix on ice (with 10% overage for pipetting error). (Table 2)</li>
<li>Gently pipette Master Mix or flick to mix, and then centrifuge briefly.</li>
<li>Transfer 80&micro;l to each sample and gently mix by pipetting up and down 3-4 times.</li>
<li>Incubate at 16&deg;C for 2 hours in thermocycler.</li>
<li>After the 2 hour incubation, proceed with the cDNA clean-up step or freeze immediately at &ndash;20&deg;C.</li>
</ol>
Part 2: Double-stranded cDNA clean-up
<ol>
<li>Remove the cDNA Pure from the refrigerator and allow it to equilibrate to room temp for 30 minutes before use. Shake the bottle to fully resuspend the magnetic cDNA binding beads before use.</li>
<li>Aliquot nuclease-free water into a 1.5mL tube and incubate at 50-60&deg;C for at least 10 minutes during the previous 2hr incubation.</li>
<li>Add 180&micro;l of cDNA Pure to each sample, and thoroughly mix by pipetting up and down. Transfer the samples to a 96-well round-bottom plate.</li>
<li>Continue to mix the samples by gently shaking the plate on an orbital shaker for at least 2 minutes.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads. Leave plate on the stand for approximately 6 minutes, or until the mixture becomes transparent and the binding beads have pelleted.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads. Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Add 150&micro;l cDNA Wash Buffer to each well and shake the plate for 1 minute on the orbital shaker at moderate speed. Beads will NOT disperse at this step, due to the low surface tension of the wash buffer.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads. Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Repeat the wash a 2nd time with 150&micro;l cDNA Wash Buffer.</li>
<li>After the 2nd wash, dry the beads by shaking the plate for 2 minutes on the orbital shaker at the maximum speed. Do not overdry!</li>
<li>Elute the cDNA from the beads by adding 18&micro;l of the preheated nuclease-free water to each sample.</li>
<li>Vigorously shake the plate for 3 minutes on the orbital shaker, then check to make sure the magnetic beads are fully dispersed. If not, continue shaking.</li>
<li>Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads.</li>
<li>Carefully transfer the eluted cDNA (~16&micro;l) to a new PCR plate (or PCR tubes).</li>
<li>Proceed directly to the next step, or freeze the cDNA at -20&deg;C. </li>
</ol>
Part 3: in vitro Transcription (IVT)
<ol>
<li>Prepare the IVT master mix at room temperature. (Table 3)</li>
<li>Gently pipette the Master Mix or flick to mix, and centrifuge briefly.</li>
<li>Add 24&micro;l to each sample, and gently mix by pipetting up and down 3-4 times.</li>
<li>Incubate at 37&deg;C for 14 hours in a thermocycler, then hold at 4&deg;C until ready for the next step.</li>
</ol>
Part 4: aRNA clean-up after IVT
<ol>
<li>Vortex the RNA binding beads briefly to obtain an even mixture before use.</li>
<li>Prepare the aRNA Binding Mix at room temperature. (Table 4) This can be done ahead of time--the prepared binding mix can be stored at room temperature for up to one week.</li>
<li>Mix well by vortexing.</li>
<li>Aliquot the aRNA Elution Buffer into a 1.5mL tube and incubate at 50-60&deg;C for at least 10 minutes.</li>
<li>Add 70&micro;l of the aRNA binding mix to each sample and mix well by pipetting up and down 3-4 times.</li>
<li>Transfer the samples from the PCR plate to a 96-well round-bottom plate.</li>
<li>Add 50&micro;l of 100% isopropanol to each sample and mix well by pipetting up and down 3-4 times.</li>
<li>Gently shake the plate on an orbital shaker for at least 2 minutes to thoroughly mix the samples.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads. Leave the plate on the stand until the mixture becomes transparent and the binding beads have pelleted.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads. Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Add 100&micro;l aRNA Wash Solution to each well and shake the plate for 1 minute on the orbital shaker at moderate speed. Beads may not fully disperse at this step.</li>
<li>Move the plate to a magnetic stand to capture the magnetic beads.</li>
<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads. Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.</li>
<li>Remove the plate from the magnetic stand.</li>
<li>Repeat the wash a 2nd time with 100&micro;l aRNA Wash Solution.</li>
<li>After the 2nd wash, dry the beads by shaking the plate for 1 minute on the orbital shaker at the maximum speed. Do not overdry the samples!</li>
<li>Elute the aRNA from the beads by adding 40&micro;l preheated aRNA Elution Buffer to each sample.</li>
<li>Vigorously shake the plate on the orbital shaker for 3 minutes, then check to make sure the magnetic beads are fully dispersed. If not, continue shaking.</li>
<li>Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads. The supernatant contains the cleaned-up amino-allyl incorporated aRNA.</li>
<li>Carefully transfer the eluted aRNA to a new PCR plate (or PCR tubes).</li>
<li>(Optional step) Check the RNA concentration of the samples by measuring 1.5&micro;l on a NanoDrop spectrophotometer.</li>
<li>Immediately continue onto the next step, or store the aRNA at &ndash;80&deg;C.</li>
</ol>
Table 1: cDNA 1st Strand Synthesis Master Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</td>
</tr>
<tr>
<td>10X First Strand Buffer</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>dNTP Mix</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>Ribonuclease Inhibitor</td>
<td>1 &micro;l</td>
</tr>
<tr>
<td>Array Script</td>
<td>1 &micro;l</td>
</tr>
</tbody>
</table>
Table 2: cDNA 2nd Strand Synthesis Master Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</td>
</tr>
<tr>
<td>Nuclease free water</td>
<td>63 &micro;l</td>
</tr>
<tr>
<td>10X Second Strand Buffer</td>
<td>10 &micro;l</td>
</tr>
<tr>
<td>dNTP Mix</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>DNA Polymerase</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>RNase H</td>
<td>1 &micro;l</td>
</tr>
</tbody>
</table>
Table 3: IVT Master Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</td>
</tr>
<tr>
<td>aaUTP solution (75 mM)</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>ATP, CTP, GTP mix (25 mM)</td>
<td>12 &micro;l</td>
</tr>
<tr>
<td>T7 UTP solution (75 mM)</td>
<td>2 &micro;l</td>
</tr>
<tr>
<td>T7 10X Reaction Buffer</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>T7 Enzyme Mix</td>
<td>4 &micro;l</td>
</tr>
</tbody>
</table>
Table 4: aRNA Binding Mix
<table border="1" cellpadding="8">
<tbody>
<tr>
<td>Reagent</td>
<td>Amount for 1 reaction</td>
</tr>
<tr>
<td>RNA Binding Beads*</td>
<td>10 &micro;l</td>
</tr>
<tr>
<td>Bead Resuspension Solution*</td>
<td>4 &micro;l</td>
</tr>
<tr>
<td>100% isopropanol**</td>
<td>6 &micro;l</td>
</tr>
<tr>
<td>aRNA Binding Buffer Concentrate</td>
<td>50 &micro;l</td>
</tr>
</tbody>
</table>
* Mix the RNA binding beads with the bead resuspension solution first. ** Add the isopropanol and mix well before adding the aRNA binding buffer concentrate.

<ol>
	<li>Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, 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.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

The authors have nothing to disclose.

Critical Steps
A second round of amplification is not advised as biases in array data have been observed. Mixing carefully at each enzymatic step (1st and 2nd strand cDNA synthesis, IVT) is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature. A PCR cycler with adjustable heated lid is preferred &ndash; even deviations as small as 2-3 degrees during IVT in an air hybridization oven or water bath hybridiza...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1753</td>
<td>For 20 reactions</td>
</tr>
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1821</td>
<td>For 100 reactions, in a 96-well format</td>
</tr>
<tr>
<td>NanoDrop ND-1000 UV-VIS spectrophotometer</td>
<td>Other</td>
<td>NanoDrop</td>
<td>ND-1000</td>
<td>Or equivalent spectrophotometer</td>
</tr>		</tbody>
		</table>
		</div>

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2 at JoVE.com

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

8.0K vues.  MIT - Massachusetts Institute of Technology. Protocole pour la vaccine infection de cellules HeLa et l'analyse de l'hôte et l'expression des gènes viraux.

Vidéo: Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...