The authors thank S-Y Choi (Chonnam National University) for valuable comments and thorough reading of our manuscript. This work was supported by a research fund of Chungnam National University.

1. Transgene Subcloning
<ol>
	<li>Choose an appropriate restriction enzyme recognition site (or sites) in the MCS of pEUI(+), and subclone the gene of interest in the desired direction (Figure 1). 
	NOTE: The EGFP or FLAG epitope-tagged ankyrin repeat domain 13A (ANKRD13A) transgene was subcloned in EcoRI site of the pEUI(+) vector using T4 DNA polymerase in a sequence- and ligation-independent cloning method19.</li>
	<li>Linearize the pEUI(+) with EcoRI for 1 h at 37 &#176;C. Mix 50 ng/&#181;L of linearized pEUI(+) with 40 ng/&#181;L of PCR-amplified EGFP or ANKRD13A, and react with T4 DNA polymerase (1 U) for 1 min at room temperature (RT) before incubation on ice for 10 min. Add 10 &#181;L of the reaction mixture directly to 100 &#181;L of DH10B competent cells for the following transformation experiment. 
	NOTE: Although several restriction enzyme recognition sites were added to the MCS of pEUI(+) (Figure 1B), we recommend subcloning the target gene into the EcoRI site, using ligation-independent cloning technology19,20.</li>
	<li>Verify the insert by sequencing and then purify the DNA using a DNA purification kit that is suitable for transfection.</li>
</ol>
2. Transient Transfection and Teb Treatment
<ol>
	<li>Prepare four cell culture dishes (60 mm diameter) containing freshly passaged HEK293 cells in 5 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 5 - 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 &#181;g/mL streptomycin). Hereafter, &#34;cell culture medium&#34; or simply &#34;culture medium&#34; represents the DMEM supplemented with FBS and antibiotics. Maintain the HEK293 cells at 37 &#176;C.</li>
	<li>Transfect 1 &#181;g of purified DNA into the HEK293 cells at 50 - 60% density using 3 &#181;L of transfection reagent. Add the transfection reagent to 200 &#181;L of diluent (DMEM without FBS and antibiotics) containing the DNA. After the incubation of the reaction mixture for 30 min at RT, add the mixture to the HEK-293 cells on the culture dish. Use two dishes for transfection, and leave the other two untreated as controls.</li>
	<li>After 12 - 24 h of transfection, add Teb (final concentrations, 0.2 -&#160;10 &#181;mol) to one of the transfected samples and to one of the non-transfected controls. Leave the other two samples untreated as experimental controls. 
	NOTE: The Teb stock solution (50 mM dissolved in dimethyl sulfoxide) can be diluted in cell culture medium, DMEM, or phosphate-buffered saline (PBS) at an appropriate concentration before adding to the cultured cells depending on the experimental parameters. However, due to the low solubility of Teb in aqueous solution, avoid preparing a master mix with a high concentration of Teb in medium; instead, replace the complete cell culture medium with fresh medium containing Teb at the desired concentration.</li>
	<li>Cultivate the Teb-treated and untreated cells for 12 - 48 h at 37 &#176;C.</li>
	<li>If using the EGFP insert, analyze EGFP expression under a fluorescent microscope ( Figure 2). Otherwise, harvest the cells for an immunoblotting assay.
	<ol>
		<li>For the immunoblotting assay, rinse the cells twice with ice-cold PBS and then scrape them out using a cell scraper.</li>
		<li>For piling the cells, spin down the harvests in a 15 mL conical tube at 21,000 x g for 2 min at 18-25 &#176;C (RT).</li>
		<li>After discarding the PBS completely, add 0.5 mL of ice-cold mammalian protein extraction reagent (M-PER), together with protease inhibitor (5 &#181;L of 100x&#160;protease inhibitor cocktail), to the pellet if the cells are cultured in a 60 mm dish.</li>
		<li>Re-suspend the cells with several rounds of pipetting, and then incubate the suspended cells for 15 min at 18 - 25 &#176;C (RT).</li>
		<li>Spin the cell lysate at 21,000 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer the aqueous supernatant into a fresh 1.5 mL microcentrifuge tube.</li>
		<li>Analyze the supernatant by immunoblotting, following standard protocols with appropriate antibodies.</li>
	</ol>
	</li>
</ol>
3. Generation of Stable HEK293 Cell Lines with Genomic Integration of the pEUI(+) Gene Switch
<ol>
	<li>Transfect 5 - 10 &#181;g of the purified pEUI(+) with the transgene into HEK293 cells that are 50 - 60% confluent in a 90 mm diameter cell culture dish, using 15 - 30 &#181;L of the transfection reagent. Add the transfection reagent to 500 &#181;L of diluent (DMEM without FBS and antibiotics) containing the DNA. After the incubation of the reaction mixture for 30 min at RT, add the mixture to HEK-293 cells on the culture dish.</li>
	<li>After the transfection, allow the transfectants to grow and express the puromycin-resistance gene products under non-selective culture medium (without puromycin) for 24 - 48 h at 37 &#176;C.</li>
	<li>Dissociate the cells from the culture dishes by trypsinization. After discarding the culture medium, rinse the transfectants with pre-warmed PBS (37 &#176;C), add 1 mL of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA), and incubate for 1 min at 37 &#176;C. After quenching the trypsin/EDTA by adding pre-warmed culture medium (10 mL, 37 &#176;C), harvest the cells using a cell scraper, followed by centrifugation in a 15 mL conical tube at 21,000 x g for 2 min at 18-25&#176;C.</li>
	<li>Re-suspend the transfectants in 50 mL of pre-warmed culture medium (37 &#176;C), and transfer 10 mL of the cells into 90 mm fresh cell culture dishes. Culture the cells at 37 &#176;C until 50 - 60% confluency before the puromycin treatment (in general, this takes approximately 24 - 48 h).</li>
	<li>Cultivate the transfectants at 37 &#176;C in cell culture medium supplemented with 3 &#181;g/mL puromycin for 3 - 4 weeks. During the selection period, change the cell culture medium containing puromycin (3 &#181;g/mL) every 2 -&#160;3 days to maintain selection pressure. When the cells become fully confluent, split them following the procedures in steps 3.3 - 3.4, with puromycin-containing culture medium. Discard the remaining cells in the culture medium, if not needed. 
	NOTE: Puromycin selection conditions must be established experimentally for specific cell types. We used 3 &#181;g/mL of puromycin in cell culture medium; this was enough to induce cell death in non-transfected HEK293.</li>
	<li>Harvest the cells from individual colonies, using a cloning cylinder, and follow the manufacturer&#39;s instructions.
	<ol>
		<li>Discard the cell culture medium. Rinse the plate twice with pre-warmed (37 &#176;C) PBS to remove floating cells.</li>
		<li>Using sterile forceps, set the cloning cylinder over the individual colonies.</li>
		<li>Add 0.2 mL of 0.35% trypsin/EDTA to each cylinder.</li>
		<li>Incubate the dish at 37 &#176;C for 1 min until the cells are detached, and then add a few drops of pre-warmed (37 &#176;C) culture medium to the cylinders.</li>
		<li>Transfer the cells from each cylinder to individual wells of a 6-well plate, with 2 mL of culture medium and 1 &#181;g/mL of puromycin in each well. 
		NOTE: When the cells become confluent, scale up the culture. Now clone(s) may be established.</li>
	</ol>
	</li>
	<li>Maintain individual clones in cell culture medium supplemented with 1 &#181;g/mL of puromycin.</li>
	<li>Validate individual colonies by testing their sensitivity to Teb treatment (final concentration, 10 &#181;M) for 24 h. Test the responsiveness of the clones to the Teb by analyzing the expression level of the transgene with immunoblotting. Select a desired clone (or clones). Cultivate a Teb-treated and an untreated sample in culture medium for 12 - 48 h at 37 &#176;C before analyzing the induction level of the transgene. The expression level of transgene can be measured through immunoblotting, following the procedures in steps 2.5.1 - 2.5.7.</li>
</ol>
4. Depletion of Teb
<ol>
	<li>Quench the Teb stimuli by replacing Teb containing cell culture medium with Teb-free medium. 
	NOTE: It is recommended to replace the growth media without first reseeding the cells onto a new culture dish. Residual Teb appears to cling to the plastic dish and will elicit a strong background expression of the transgene. Before reseeding the cells on a new culture plate, rinse the harvested cells several times with either culture medium or PBS.
	<ol>
		<li>Dissociate the cells from the culture dishes by trypsinization. After discarding the Teb containing culture medium, rinse the Teb-stimulated cells twice with pre-warmed PBS (37 &#176;C), add 1 mL of 0.25% trypsin/EDTA, and incubate for 1 min at 37 &#176;C. After quenching the trypsin/EDTA by adding pre-warmed Teb-free culture medium (10 mL, 37 &#176;C), harvest the cells using a cell scraper, followed by centrifugation in a 15 mL conical tube at 21,000 x g for 2 min at 18-25 &#176;C.</li>
		<li>Re-suspend the cells in 50 mL of pre-warmed Teb-free culture medium (37 &#176;C), and transfer 5 mL of the cells into three 60 mm fresh cell culture dishes. Culture the cells at 37 &#176;C for 24, 48, and 72 h respectively.</li>
		<li>Harvest the cells at different time points and measure the expression level of the transgene by using immunoblotting. 
		NOTE: Under these experimental conditions, the transgene expression became undetectable approximately 3 days post-culture of the cells in the Teb-free cultural medium (Figure 3). The expression level of transgene can be measured by immunoblotting, as per steps 2.5.1 - 2.5.7.</li>
	</ol>
	</li>
</ol>

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We have no conflicts of interest related to this report.

The main drawback of using a binary gene expression switch is the need to simultaneously deliver two separate plasmids (driver and effector) into the target cell. This can lead to an unequal distribution of plasmids and result in an inconsistent response of the switch to the inducers1,2,3. Another shortcoming of the two-vector system is that a minimum of two rounds of antibiotic selection are required to identify promising cell ...

Several different gene switches have been explored for their ability to precisely regulate transgene expression in cell culture and animal models, with varying degrees of success1,2,3. Potential gene switch systems should meet several stringent criteria, including: precise directional expression of the transgene, dosage- and time-dependent responsiveness to inducers, negligible leakiness of the promoter, reversibility of transgene activation through inducer removal, and inducer toxicity levels tolerable to cell lines and laboratory animals1,2,3. Several small molecule inducers have been exploited, including tetracycline4,5, rapamycin6,7,8, mifepristone9,10, and ecdysone11,12,13,14. In general, these systems require at least two plasmids containing two or three discrete expression units, one or two of which compose a regulatory element (inducer plasmid) that binds a small molecule inducer to gain transcriptional activity; and another plasmid (effector plasmid) containing the transgene under the control of a regulatory DNA region that allows the access of the inducer-bound regulatory element. The main drawback of the binary system is that it requires the concomitant introduction of two vectors (inducer and effector) into the target cell. In transient transgene expression experiments, the simultaneous transfection of two plasmids inevitably produces a population of cells that is singly transfected with either the inducer or the effector, or co-transfected unequally. In addition, the binary system requires at least two rounds of antibiotic selection to establish stable cell lines, which is time-consuming and laborious. Thus, combining all of the components required for transgene expression and regulation into a single vector would be ideal to guarantee the concomitant expression of all the required elements in a single cell and allow for the regulation of transgene expression through treatment with small molecule inducers.
To overcome the shortcomings of binary gene switches, we recently developed a singular gene switch, using a Drosophila melanogaster EcR-based gene inducible system15. Ecdysone mediates gene expression when it binds to the EcR/ultraspiracle (USP) heterodimer, which in turn induces binding of the EcR to DNA regulatory elements3,11. The vertebrate retinoid X receptor (RXR), an ortholog of insect USP, recruits EcR to form an active transcriptional activator16. Thus, for the targeted expression of a transgene in vertebrate cells or tissue, the EcR and RXR must be co-expressed simultaneously before being stimulated by ecdysone or its agonists. Since the heterodimeric feature of the EcR-based gene switch can be influenced by the endogenous RXR level, Padidam et al. replaced the EcR DNA-binding and activation domains with heterologous GAL4 DNA-binding, herpes simplex virus protein vmw65 (VP16) activation domains fused to the EcR ligand-binding domain, which then became unresponsive to endogenous RXR and formed a homodimer to gain transcriptional activity17. The EcR-based gene switch was further improved by constructing a chimeric protein comprised of minimal VP16 activation domain combined with GvEcR, which formed a homodimer and localized in the cytosol in the absence of the ecdysone agonist, Teb16,18. By binding to Teb, the GvEcR changed its subcellular localization from the cytosol to the nucleus to recognize a hybrid promoter comprised of zebrafish E1b minimal promoter combined with a ten tandem repeated upstream activation sequences (UASs) to initiate the transcription of the target gene16.
To simplify the previously reported EcR-based gene switches16,18, we combined the binary features of the system into a single vector equipped with all the required elements for stimulating transgene expression, and then designated the newly generated vector, pEUI(+) (GenBank accession number: KP123436, Figure 1A)15. The effector responding to the GvEcR driver (hereafter driver) consists of ten tandem UAS repeats next to an E1b minimal promoter followed by a multiple cloning site (MCS) with EcoRI, PmlI, NheI, BmtI, AfeI, and AflII recognition sequences (Figure 1B). Additionally, to facilitate the selection of transfected cells, an SV40 promoter-driven puromycin N-acetyl-transferase (PAC) gene was placed between the driver and effector regions to maintain stable cell lines (Figure 1).
We describe a detailed protocol for the transient and stable modulation of transgene expression using pEUI(+). In addition, we provide detailed instructions for the successful use of Teb as an ecdysone agonist.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pEUI(+)</td>
			<td style="width:71px;">TransLab</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The patent license was transferred to TransLab Inc.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gene-Fect Transfection Reagent</td>
			<td style="width:71px;">TransLab</td>
			<td style="width:119px;">TLC-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tebufenozide</td>
			<td style="width:71px;">Fluka</td>
			<td style="width:119px;">31652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cloning cylinder</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CLS31668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">58-58-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">LM001-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">S001-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture dish</td>
			<td style="width:71px;">SPL life science</td>
			<td style="width:119px;">11090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse FLAG M2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F3165</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-alpha tubulin antibody</td>
			<td style="width:71px;">Calbiochem</td>
			<td style="width:119px;">CP06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cloning cylinder</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CLS31668</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoBond Xtra Midi</td>
			<td style="width:71px;">Macherey-Nagel</td>
			<td style="width:119px;">740410.1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M-PER Mammalian Protein 
			Extraction Reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">78505</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X Protease inhibitor Cock. III</td>
			<td style="width:71px;">T&#38;I</td>
			<td style="width:119px;">BPI-9200</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ecdysone receptor-based singular gene switch for deliberate expression of transgene with robustness, reversibility, and negligible leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

The GvEcR-based singular gene switch is depicted in Figure 1A. The pEUI(+) vector was optimized for regulated transgene expression by the treatment with Teb. The effector region of pEUI(+) comprises a 10xUAS and an E1b minimal promoter followed by an MCS containing EcoRI, PmlI, NheI, BmtI, AfeI, and AflII restriction enzyme recognition sites. An SV40 poly(A) signal was added behind the MCS (Figure 1B). A PAC gene was ins...

Watch this Scientific Journal Video about An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness at JoVE.com

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

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Research

JoVE Journal

Genetics

6.4K Views.  Chungnam National University. Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment. 

Video: An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...