This work was supported in part by the National Science Foundation (IOS-0920417) (ACL).

1. Construction of Lentiviral Luciferase Reporters
A mammalian circadian reporter construct usually contains an expression cassette in which a circadian promoter is fused with the luciferase gene. Both ligation- and recombination-based strategies are commonly used for DNA cloning. As an example, here we describe a recombination-based Gateway cloning method for generating a P(Per2)-dLuc lentiviral reporter, in which the destabilized luciferase (dLuc) is under control of the mouse Per2 promoter.
<ol>
	<li>Cloning of Per2 promoter. Use PCR to amplify the Per2 promoter DNA fragment of 526 bp, upstream of the transcription start site from a mouse Per2 BAC clone9-13, using a forward primer (5'-CTCGAGCGGATTACCGAGGCTGGTCACG TC-3') and a reverse primer (5'-CTCGAGTCCCTTGCTCGGCCCGTCAC TTGG-3'), and clone into pENTR5'-TOPO vector (Invitrogen) to generate pENTR5'-P(Per2).</li>
	<li>Cloning of dLuc. The dLuc contains the firefly luciferase gene and a C-terminal PEST sequence for rapid protein degradation as previously described21. Use PCR to amplify the dLuc DNA fragment, and clone into pENTR/D-TOPO vector (Invitrogen) to generate pENTR/D-dLuc.</li>
	<li>Construction of reporter vector. Mix the two pENTR plasmids, pENTR5'-P(Per2) and pENTR/D-dLuc, with the lentiviral destination vector pLV7-Bsd (Bsd, blasticidin resistance gene), and perform the recombination reaction using Clonase to generate a pLV7-Bsd-P(Per2)-dLuc reporter (Figure 1). pLV7-Bsd is a modified version (made in our lab) of pLenti6/R4R2/V5-DEST (Invitrogen) in which the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequences22 were inserted immediately downstream of the expression cassette to enhance gene expression.</li>
</ol>
2. Production of Lentiviral Particles
1. Seed 293T cells (day 1)
<ol>
	<li>Grow human embryonic kidney (HEK) 293T cells to 90-100% confluence in regular DMEM supplemented with 10% FBS and 1x Penicillin-Streptomycin-Glutamine (PSG) on 10 cm culture dishes. (Rapidly growing cells with low passage number are critical for efficient transfection.)</li>
	<li>Prior to seeding the cells for transfection, coat 6-well culture plates by adding 1 ml of 0.001% poly-L-lysine in PBS to each well and incubate at room temperature for 20 min. Aspirate the solution and rinse once with 1x PBS before use.</li>
	<li>Dissociate 293T cells with trypsin and seed 0.75 x 106 cells onto each well of the pre-coated plates with 2 ml regular DMEM. Swirl the plates thoroughly to obtain an even distribution of cells in each well. Grow the cells in the incubator at 37 &deg;C overnight.</li>
</ol>
2. Transient transfection via CaPO4/DNA precipitation (day 2)
<ol>
	<li>Observe the seeded cells from day 1. Cell should reach confluence of 80-90%.</li>
	<li>Prepare plasmid transfection mix in a 1.5 ml microcentrifuge tube by adding 2 &mu;g of a lentiviral reporter plasmid DNA (e.g., pLV7-P(Per2)-dLuc; Liu lab) and the 3 packaging vectors (1.3 &mu;g Gag/Pol, 0.5 &mu;g Rev, and 0.7 &mu;g VSVG; Invitrogen). As a control for both transfection and subsequent infection, we usually include an additional well in transfection for a lentiviral GFP expression vector, pLV156-CMV-EGFP (Figure 1A), harboring enhanced green fluorescent protein (EGFP) under the control of the CMV promoter as described previously20.</li>
	<li>Add 100 &mu;l of 0.25 M CaCl2 (diluted with DNase/RNase-free ddH2O from 2.5 M stock) to the plasmid mix in step 2 and mix thoroughly. Then add 100 &mu;l of 2x BBS solution (50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 6.95) and mix gently but thoroughly. Incubate the DNA mix at room temperature for 15 min.</li>
	<li>While waiting, aspirate medium from 293T cells and change to 2 ml fresh medium. Return the plate to the incubator for at least 10 min to equilibrate medium pH before transfection.</li>
	<li>Add transfection mix from step 3 to 293T cells drop by drop. Swirl the plate gently and observe particle formation under a microscope. Incubate at 5% CO2, 37 &deg;C overnight. (Fine particle formation of CaPO4/DNA precipitate is critical for efficient transfection.)</li>
</ol>
3. Harvest viral particles (days 3-4)
<ol>
	<li>About 16 hours post-transfection (day 3) by which time cells should reach 100% confluence, aspirate medium from cells and replace with 2 ml fresh regular DMEM. Incubate at 37 &deg;C overnight.</li>
	<li>On day 4, assess transfection efficiency by observing EGFP expression in transfection control cells (Transfection efficiency of 90-100% with high EGFP expression is a reliable predictor of a good viral prep.)</li>
	<li>Collect the medium containing secreted, infectious viral particles. Centrifuge at &gt;2,000 x g for 5 min to remove residual 293T cells and collect the virus-containing supernatant. Alternatively, the medium may be cleared with a 0.45 &mu;m membrane filter. The viral particles are ready for use in infection.</li>
</ol>
3. Infection of 3T3 Cells
1. Seed 3T3 cells (day 3)
Split and seed appropriate number (~12,000) of 3T3 cells on a 12-well plate to obtain 20-30% confluence by next day. Incubate at 37 &deg;C overnight.
2. Infect 3T3 cells (day 4)
<ol>
	<li>Observe the seeded cells. Confluence of 20-30% (less than 50%) is desired for infection.</li>
	<li>Add polybrene to a final concentration of 5 &mu;g/ml to the collected medium containing viral particles. Mix well by pipetting.</li>
	<li>Aspirate medium from 3T3 cells, and add 1 ml of the above viral mixture per well. Incubate at 37 &deg;C overnight. (Polybrene is used to enhance infection efficiency, but is not absolutely required. As it may be toxic to some cells, prior testing is recommended.)</li>
</ol>
3. Select infected cells (day 5 and onward)
<ol>
	<li>Twenty-four hours post-infection, aspirate medium containing virus and polybrene from infected cells, wash once with 1x PBS, and change to fresh medium. Incubate at 37 &deg;C overnight for recovery and growth.</li>
	<li>When confluent (usually 1-2 days later), split the cells and incubate at 37 &deg;C overnight.</li>
	<li>The following day, aspirate medium from cells (&lt;50% confluence is desired) and replace with fresh medium containing 10 &mu;g/ml Blasticidin to select for stably transduced cells. (Blasticidin kill-curve needs to be empirically determined for a particular cell line.)</li>
	<li>Change to fresh medium containing Blasticidin every 2-3 days for continuous selection of antibiotic-resistant cells expressing clock reporters (generally 4-6 days total).</li>
</ol>
4. Bioluminescence Recording of Reporter Cells
1. Seed reporter cells
Propagate Blasticidin-resistant reporter cells and split onto 35-mm culture dishes. Incubate at 37 &deg;C until confluent. We usually prepare &ge;3 dishes for each reporter cell line under each condition for circadian phenotyping.
2. Synchronization and change to recording medium
<ol>
	<li>Aspirate medium from confluent reporter cells, wash once with PBS, and replace with DMEM containing 10 &mu;M forskolin (or 200 nM dexamethasone). Incubate at 37 &deg;C for 1 hour to synchronize the cells. (Alternatively, cells can be synchronized by temperature cycles23 or serum shock24.)</li>
	<li>While waiting, prepare recording medium for 3T3 cells as follows: 1x DMEM (HyClone) containing 10% FBS, 1x Pen/Strep/Gln, 1 &mu;M forskolin, 1 mM luciferin, 25 mM HEPES, pH 7.4. Serum and forskolin concentration may be determined empirically. For very dim cells, phenol red-free medium may be used.</li>
	<li>At the end of forskolin treatment, aspirate medium and replace with freshly made recording medium.</li>
</ol>
3. Bioluminescence recording of reporter cells
<ol>
	<li>Following medium change, cover culture dishes with 40 mm sterile coverslips and seal in place with vacuum grease to prevent evaporation.</li>
	<li>Load the dishes onto the LumiCycle luminometer, which is kept inside an incubator set at 36 &deg;C without H2O or CO2.</li>
	<li>Start real-time bioluminescence recording. We usually record rhythms for 1 week, followed by medium change and continuous recording for a second week (see Savelyev et al. for details)25. (For recording of 96-well plates, Synergy SL2 was used as the recording device; see Discussion 1.1 for detail.)</li>
</ol>
5. Data Analysis and Presentation
Reporter cells facilitate high-resolution quantitative luminescence recording, critical for determining phenotypic effects on circadian clock function. To obtain circadian parameters including phase, period length, rhythm amplitude, and damping rate, we use the LumiCycle Analysis program (Actimetrics) to analyze bioluminescence data5,14. Briefly, raw data are baseline fitted first, and baseline-subtracted data are fitted to a sine wave, from which the parameters are determined. For samples that show persistent rhythms, goodness-of-fit of &gt;90% is usually achieved. Due to high transient bioluminescence upon medium change, we usually exclude the first cycle of data from analysis.
For data presentation, we usually plot raw data (bioluminescence, counts/sec) against time (days). When necessary, baseline-subtracted data can be plotted to compare amplitude and phase.
6. Representative Results
1. Phase-specific circadian reporters
The circadian clock is based on a biochemical negative feedback mechanism1. The core feedback loop consists of transcriptional activators BMAL1 and CLOCK, and repressors PERs and CRYs, which act on the circadian E/E'-box enhancer elements to produce rhythmic gene expression (with morning phase, e.g., Rev-erb&alpha;). The core loop regulates and integrates at least two other circadian cis-elements, the DBP/E4BP4 binding element (D-box; for day phase, e.g., Per3) and the ROR/REV-ERB binding element (RRE; for night phase, e.g., Bmal1)17. Combinatorial regulation by multiple circadian elements can generate novel intermediate phases. For example, Cry1 transcription is mediated by all three circadian elements (i.e., E/E'-box and D-box elements in the promoter and RREs in the first intron of the Cry1 gene), giving rise to the distinct Cry1 evening-time phase13.
Based on these mechanisms of gene regulation, we generated four different reporter constructs: P(Per2)-dLuc and P(Cry1)-dLuc reporters containing both E/E'-box and D-box elements in the regulatory region17,26,27; P(Cry1)-Intron-dLuc representing combinatorial regulation by all three elements (i.e., E/E'-box, D-box, and RRE)13,17; and P(Bmal1)-dLuc regulated exclusively by RRE9,17,19,21. We introduced these reporters into 3T3 cells to produce the anticipated distinct phases of reporter expression (Figure 2).
2. Gene knockdown via RNAi and pharmacologically active compounds
When transfection efficiency is high, synthetic siRNA can be transiently transfected into cells to knock down gene expression. When transfection is technically difficult, an shRNA expression vector can be stably transduced into cells via lentiviral infection, so that shRNA produced by the cell is processed to siRNA for gene knockdown (KD). Here we present KD effects of Cry1 and Cry2 genes using siRNA in U2OS cells (Figure 3A) and shRNA in 3T3 cells using a pLL3.7 Gateway expression vector9 (Figure 3B). In addition to 3T3 cells, the U2OS model has become another preeminent cellular clock model largely because it meets the key requirements for high-throughput screening of commercially available human siRNA libraries (e.g., human origin, capable of generating robust circadian rhythms, validated function of all known clock genes, and amenable to highly efficient transfection and quantitative luminescence recording). In both cell types, RNAi-mediated KD resulted in clock phenotypes consistent with previous mouse knockout (KO) and cellular KD studies5,10,11,28. For example, Cry1 KD shortens period length and reduces rhythm persistence, whereas Cry2 KD lengthens period. In addition, selected small molecules can be used to pharmacologically target and perturb protein function (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/4234/4234fig1.jpg" /> 
Figure 1. The lentivirus-mediated gene delivery system. (A) Schematic diagram of two lentiviral P(Per2)-dLuc reporter vectors and a CMV-EGFP construct. Only the region for integration into the host cell genome is shown. In both reporter constructs, the transcription of dLuc is under direct control of the Per2 promoter. In the pLV7-Bsd-P(Per2)-dLuc vector (recombination-based cloning), a coexpressed Blasticidin resistance gene (Bsd) facilitates selection of infected cells. In the pLV156-P(Per2)-dLuc vector (ligation-based cloning), EGFP translation is mediated by an internal ribosome entry site (IRES) downstream of dLuc, allowing for visual observation and FACS sorting of infected cells. In addition, an SV40 promoter/terminator (P/T) is used as an insulator (see discussion 1.3). In the CMV-EGFP control vector, EGFP expression is under control of a strong CMV promoter. (B) Fluorescent images of transfected and infected GFP-expressing cells. Typically, we achieve high efficiency both in transient transfection of 293T cells and in lentiviral infection of cell lines of our interest, as indicated by GFP expression in these cells. <a href="https://www.jove.com/files/ftp_upload/4234/4234fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4234/4234fig2.jpg" /> 
Figure 2. Phase-specific expression of bioluminescence reporters in 3T3 cells. The lentiviral reporter vectors used in this experiment are pLV7-Bsd-P(Per2)-dLuc, P(Cry1)-dLuc, P(Cry1)-Intron-dLuc, and P(Bmal1)-dLuc. Each reporter exhibits a distinct phase of oscillation, as indicated by the arrows. While the Per2 and Cry1 promoters drive peak bioluminescence at morning-day phases and the Bmal1 promoter at night phase, combinatorial regulation by the P(Cry1)-Intron harboring E-box, D-box, and RRE elements confers evening phase of peak bioluminescence. <a href="https://www.jove.com/files/ftp_upload/4234/4234fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4234/4234fig3.jpg" /> 
Figure 3. Genetic and pharmacological perturbation of circadian bioluminescence rhythms in reporter cells. (A) Effects of Cry1 and Cry2 knockdown by siRNAs on cellular rhythms of U2OS reporter cells. The LumiCycle luminometer was used for bioluminescence recording of cells in 35 mm dishes. Figure is adapted from Reference #10, with permission from Elsevier (2009). (B) Effects of Cry2 knockdown by shRNAs on cellular rhythms of 3T3 reporter cells. A pLL3.7 Gateway vector containing a U6-shRNA cassette was used for Cry2 gene knockdown. shRNA2 has a better knockdown efficiency than shRNA1 as determined by Western blot analysis (data not shown). A Synergy luminometer was used for bioluminescence recording of cells in a 96-well plate. The settings for recording are as follows: incubator temperature, 33 &deg;C; integration time, 15 sec; interval time, 30 min. (C) Effects of small molecule inhibitors on cellular rhythms of U2OS reporter cells. Chir99021 and Roscovitine are inhibitors directed against GSK-3 and CDK, respectively. The ViewLux system (Chir99021 assay) and a Tecan luminometer (Roscovitine assay) were used for bioluminescence recordings of cells in 384-well plates. Figure is adapted from Reference #19 (Copyright 2008 National Academy of Sciences, U.S.A.). <a href="https://www.jove.com/files/ftp_upload/4234/4234fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

No conflicts of interest declared.

1. Modifications to Current Protocol
1.1 Recording devices and throughput considerations
Because of its commercial availability, the LumiCycle (Actimetrics) has become the most commonly used automated luminometer device for real-time recording4,5,9,19,29-31. The LumiCycle employs photomultiplier tubes (PMTs) as light detectors, which provide extremely high sensitivity and low noise14, and therefore is particularly suitable for data acquisition of ex...

<table border="1">
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>HyClone</td>
 <td>SH30243FS </td>
 <td>For regular cell growth</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>12100-046</td>
 <td>For luminometry</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>HyClone</td>
 <td>SH3091003</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pen/Strep/Gln(100x)</td>
 <td>HyClone</td>
 <td>SV3008201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B-27</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Luciferin</td>
 <td>Biosynth</td>
 <td>L-8220</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-L-lysine</td>
 <td>Sigma</td>
 <td>P4707 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polybrene</td>
 <td>Millipore </td>
 <td>TR-1003-G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Forskolin</td>
 <td>Sigma</td>
 <td>F6886</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>All other chemicals</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tissue culture incubator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>5% CO2 at 37&deg;C</td>
 </tr>
 <tr>
 <td>Tissue culture hood</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>BSL-2 certified</td>
 </tr>
 <tr>
 <td>Light &amp; fluorescent microscope</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Phase contrast optional</td>
 </tr>
 <tr>
 <td>LumiCycle</td>
 <td>Actimetrics</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>

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.

Watch this Scientific Journal Video about Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters at JoVE.com

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

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.

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

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22.1K Views.  The University of Memphis. 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.

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

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 ...

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 ...

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 ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

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.

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

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...