We are grateful to our colleagues from the University of Geneva: Jacques Philippe for constructive comments on this work, Ueli Schibler for invaluable help with the development of the perifusion system and for scientific inspiration, Andr&#233; Liani for the having conceived design, manufacturing and commissioning of the perfusion system, Lesa-Technology LTD company for the assistance in the perifusion system and Drip-biolumicorder software development, George Severi for assistance with the perifusion experiments, Ursula Loizides-Mangold for critically reading the manuscript, and Anne-Marie Makhlouf for lentivirus preparations; to Etienne Lefai, St&#233;phanie Chanon and Hubert Vidal (INSERM, Lyon) for preparing human primary myoblasts; and to Domenico Bosco and Thierry Berney (Human Islet Transplantation Center, Geneva University Hospital) for providing human islets. This work was funded by the Swiss National Science Foundation Grant No. 31003A_146475/1, the Sinergia Swiss National Science Foundation Grant No. CRSII3-154405, Fondation Romande pour la Recherche sur Diab&#232;te, Bo Hjelt Foundation, Fondation Ernst et Lucie Schmidheiny, and Soci&#233;t&#233; Acad&#233;mique de Gen&#232;ve (CD).

Ethics statement: Manipulations included in this protocol were approved by the Ethics Committee of the Geneva University Hospital and by the Ethical Committee SUD EST IV (Agreement 12/111) 19. Human islets were isolated from pancreases of brain-dead multi-organ donors in the Islet Transplantation Centre at the University Hospital of Geneva (Switzerland) as described by us in references 16,18, or obtained from a commercial source.
1. Preparation of Primary Cell Culture
<...

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The experimental settings described here are composed of lentiviral delivery of circadian bioluminescence reporters into cultured human primary cells, followed by subsequent in vitro synchronization and continuous recording of bioluminescence for several days, and parallel analysis of hormone secretion by the same cells. They represent an efficient approach for exploring molecular mechanisms and functional aspects of circadian clocks in human primary cells.
The quality of the donor ma...

The circadian timing system (from Latin &#34;Circa diem&#34;) has emerged in all light-sensitive organisms, as an adaptive mechanism to the rotation of the Earth. In mammals, it is organized in a hierarchical manner, encompassing the central clock, which is situated in the suprachiasmatic nucleus of the ventral hypothalamus, and peripheral (or slave) oscillators that are operative in different organs. Moreover, these cell autonomous self-sustained oscillators are functional in nearly every cell of the body 1. Photic signals represent a dominant synchronizing cue (Zeitgeber) for the SCN neurons, whereas neural and humoral signals emanating from the SCN reset the peripheral clocks. In addition rest-activity rhythms, that drive in turn feeding-fasting cycles, are further synchronizers for peripheral clocks 2. According to our current understanding, the molecular makeup of the core clock is based on transcriptional and translational feedback loops, which are conserved between organisms. This comprises the transcriptional activators BMAL1 and CLOCK, which together activate transcription of the negative core clock PER and CRY genes. High levels of PER and CRY proteins will inhibit their own transcription through inhibition of the BMAL1/CLOCK complex. An auxiliary loop consists of the nuclear receptors REV-ERBs and RORs, which also regulate the transcription of BMAL1 and CLOCK. Furthermore, posttranslational events including phosphorylation, sumoylation, acetylation, O-GlcNAcylation, degradation and nuclear entry of the core clock proteins represent an additional important regulatory layer in establishing the 24 hr oscillation cycle 3.
Accumulating evidence stems from studies in rodent models and highlights the critical role of the circadian system in the coordination of metabolic and endocrine functions 4-5. A number of large-scale transcriptome analysis suggest that feeding &#8211; fasting cycles play a central role in the synchronization of peripheral oscillators 6-8. In an agreement with these studies, metabolomic and lipidomic analysis in rodents and humans have revealed that a large number of metabolites oscillate in tissue, plasma, and saliva in a circadian manner 9-11. Importantly, most hormones exhibit circadian rhythms in blood 5,12-13. Moreover, circadian clocks of the corresponding hormone producing peripheral tissue might regulate hormone secretion locally. Cell-autonomous circadian oscillators have been described in rodent and human pancreatic islet cells 14-16. These oscillators play an essential role in regulating the pancreatic islet transcriptome and function 15,17-18. Furthermore, myokine secretion by human skeletal myotubes has been recently demonstrated to exhibit a circadian pattern, which is regulated by cell-autonomous oscillators operative in these cells 19.
Several approaches for studying circadian rhythms in humans in vivo have been widely used. For instance, plasma melatonin or cortisol levels as well as thoracic skin surface temperature (reviewed in references 3,20) have been studied to assess endogenous circadian clocks. Although these methods allow studying systemic circadian oscillations in vivo, they are far from providing a reliable assessment of free-running autonomous circadian rhythms in different organs and tissues. Nevertheless, such dissection from the systemic regulation would be an indispensable tool for understanding the specific effect of intracellular molecular clocks on the function of these cells. Therefore, a substantial effort has been undertaken to develop reliable approaches for studying human clocks in immortalized or primary cultured cells synchronized in vitro. Importantly, it has been demonstrated that clock characteristics measured in cultured primary skin fibroblast cells closely reflect the individual clock properties of the whole organism 21. The development of fluorescent and bioluminescent circadian reporters has greatly advanced this approach 22-27. Furthermore, studying primary cell clocks that are derived from different peripheral organs allows for the investigation of the molecular properties of human tissue-specific clocks 3,5,16,19-20,28. Thus, assessment of circadian clocks in in vitro synchronized primary explants or cells, by using bioluminescent reporters, represents a highly useful method to study the molecular makeup of human peripheral clocks and their impact on organ function.
In this article, we will present detailed protocols for assessing circadian gene expression in human primary islet and skeletal muscle cells synchronized in vitro as well as the impact of autonomous cellular clock disruption on the secretory function of these cells.

<table border="0" cellpadding="0" cellspacing="0" width="1006">
	<tbody>
		<tr height="19">
			<td height="19" style="height:20px;width:215px;">Trypsin-EDTA</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">25300-054</td>
			<td style="width:560px;">For muscle biopsy digestion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DPBS no calcium no magnesium</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HAM F-10</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">41550-021</td>
			<td>For myoblasts culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FBS</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">10270</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicillin-Streptomycin</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">P0781-100</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Gentamycin</td>
			<td style="width:112px;">Axon&#160;</td>
			<td style="width:119px;">A1492.0001</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fungizone</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">15290-026</td>
			<td>Amphotericin B, supplement to culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DMEM 1g/L glucose + Na pyruvate + glutamax&#160;</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">21885-025</td>
			<td>For myotubes culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">DMEM 1g/L glucose -Na Pyruvate - glutamax</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">11880-028</td>
			<td>Recording medium for LumiCycle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glutamax</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">35050-028</td>
			<td>L-alanyl-L-glutamine dipeptide, supplement to recording medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Accutase</td>
			<td style="width:112px;">Innovative Cell Technologies</td>
			<td style="width:119px;">AT-104</td>
			<td>Cell detachment solution, for islet cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CMRL</td>
			<td style="width:112px;">Gibco</td>
			<td style="width:119px;">21530-027</td>
			<td>Culture medium for islet cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium Pyruvate</td>
			<td style="width:112px;">Gibco</td>
			<td style="width:119px;">11360-039</td>
			<td>Supplement to culture medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;width:215px;">15 ml High-Clarity Polipropylene Conical Tube</td>
			<td style="width:112px;">Falcon</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">F75 flask</td>
			<td style="width:112px;">BD Falcon</td>
			<td style="width:119px;">353136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">3.5 cm Petri dish&#160;</td>
			<td style="width:112px;">BD Falcon</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Foskolin</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">F6886</td>
			<td>Adenylyl cyclase activator, used for synchronization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Luciferin</td>
			<td style="width:112px;">Prolume LTD</td>
			<td style="width:119px;">260150</td>
			<td>Supplement to recording medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">OptiMEM&#160;</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">51985-026</td>
			<td>Serum-free Minimal Essential Medium (MEM) used for human islet cells transfection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Lipofectamine RNAiMAX reagent</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">13778-150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HiPerFect reagent</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">301705</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ON-TARGET plus siCLOCK smartpool&#160;</td>
			<td style="width:112px;">Dharmacon</td>
			<td style="width:119px;">L-008212-00</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ON-TARGET plus non targeting siRNA #1 (siControl)</td>
			<td style="width:112px;">Dharmacon</td>
			<td style="width:119px;">D-001810-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DNeasy Blood &#38; Tissue Kit&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">69504</td>
			<td>For myotubes DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RNeasy Plus Mini kit&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td>For myotubes RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">QIAshredder&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">79654</td>
			<td>For myotubes RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">2 ml collecting tubes</td>
			<td style="width:112px;">Axygen</td>
			<td style="width:119px;">311-10-051</td>
			<td>To collect the medium with the perifusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tissue culture Plate, 6 Well</td>
			<td style="width:112px;">BD Falcon&#160;</td>
			<td style="width:119px;">353046</td>
			<td>To collect the medium with the perifusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RNeasy Plus Micro kit&#160;</td>
			<td style="width:112px;">Qiagen</td>
			<td style="width:119px;">74034</td>
			<td>For islet RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Human IL-6 Instant ELISA kit&#160;</td>
			<td style="width:112px;">eBioscience</td>
			<td style="width:119px;">88-7066-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Human Insulin Kit Mercodia</td>
			<td style="width:112px;">Mercodia</td>
			<td style="width:119px;">10-1113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hydrochloric acid, min,37%,p.a.</td>
			<td style="width:112px;">Acros organics</td>
			<td style="width:119px;">124630010</td>
			<td>Used for preparation of lysis buffer (375ml Ethanol+7.5%HCl+117.5%H2O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol (&#62;99.8%)</td>
			<td style="width:112px;">Fluka Analytical</td>
			<td style="width:119px;">02860-1L</td>
			<td>Used for preparation of lysis buffer (375ml Ethanol+7.5%HCl+117.5%H2O)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Islets for Research</td>
			<td colspan="2">Prodo Laboratories</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name</td>
			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Equipment:</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Centrifuge</td>
			<td style="width:112px;">Heraeus</td>
			<td style="width:119px;">Megafuge 1.0R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:112px;">VWR</td>
			<td style="width:119px;">1112A</td>
			<td>&#160;at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tissu culture hood</td>
			<td style="width:112px;">Faster&#160;</td>
			<td style="width:119px;">SafeFastElite</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">Tissu culture incubator</td>
			<td style="width:112px;">Heraeus</td>
			<td style="width:119px;">HeraCell 150</td>
			<td style="width:560px;">5% CO2 at 37 &#176;C, no water due to the LumiCycle installation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">Tissu culture incubator</td>
			<td style="width:112px;">Heraeus</td>
			<td style="width:119px;">HeraCell 150</td>
			<td style="width:560px;">5% CO2 at 37 &#176;C, no water due to the LumiCycle installation</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:215px;">Tissu culture incubator</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td style="width:119px;">Hera Cell 150i</td>
			<td style="width:560px;">5% CO2 at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Shaker</td>
			<td style="width:112px;">Heidolph Instruments</td>
			<td style="width:119px;">Unimax 1010</td>
			<td>For agitation of the siRNA mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">LumiCycle</td>
			<td style="width:112px;">Actimetrics</td>
			<td style="width:119px;"></td>
			<td style="width:560px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">LumiCycle software</td>
			<td style="width:112px;">Actimetrics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CosinorJ software</td>
			<td style="width:112px;">EPFL</td>
			<td style="width:119px;"></td>
			<td>Freely available at: http://bigwww.epfl.ch/algorithms/cosinorj/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheodyne titan MX&#160;</td>
			<td style="width:112px;">ERC GmbH</td>
			<td style="width:119px;"></td>
			<td>Control software that controls the timing of the automated switch</td>
		</tr>
	</tbody>
</table>

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.

Assessment of Islet Hormone Secretion with Parallel Circadian Bioluminescence Recording from Perifused Human Islet Cells
After providing a first molecular characterization of the circadian clock, operative in human islet cells 16, we aimed at studying the impact of clock disruption on islet function and transcription 18. We set up an efficient siClock transfection protocol in dispersed human islet ...

Watch this Scientific Journal Video about Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures at JoVE.com

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

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 ...

The overall goal of this procedure is the parallel assessment of circadian bioluminescence and the secretory activity of human primary endocrine cells cultured and synchronized en vitro.This method can help to investigate the temporal pattern of cell secretion and how it can be regulated by cell-autonomous circadian clocks.The main advantage of this technique is that it allows us to monitor the circadian luciferins as reporter activity and, simultaneously collect outflow medium for measuring the levels of secreted hormones and myokines.To introduce circadian bioluminescence reporters into primary cells by lentiviral transduction, first, transfer the 3.5 cm petri dishes containing human myoblasts at 30%to 50%confluency to a laminar flow cabinet.Replace the human myoblast medium with 2 mL of growth medium.Then transduce the primary cell culture by pipetting lentivirus solution into the dish to obtain a multiplicity of infection equal to three.Incubate the cells overnight in a tissue-culture incubator.Change medium the next day.To achieve en vitro synchronization, add 10 micromolar adenylyl cyclase activator in 2 mL of medium per 3.5 cm petri dish containing the previously transfected primary cells.Incubate the cells for 60 minutes at 37

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Research

JoVE Journal

Genetics

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 ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

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 ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

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 ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

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 ...