We thank J. Birchler, E. Bach and the Bloomington Drosophila Stock Center for various Drosophila strains; the National Cancer Institute (NCI) Developmental Therapeutics Program for the Oncology Set small-molecule drug library; UCSD undergraduate students including Amy Chang, Taesik You, Jessica Singh-Banga, Rachel Meza, and Alex Chavez. Research reported in this publication was supported by a research grant from American Thoracic Society to J.L. and funding from NIH: R01GM131044 to W.X.L.

1. Drug library preparation
<ol>
	<li>Identify and prepare a drug library to be screened using appropriate solvent at a desired concentration (e.g., 10 mM in DMSO). 
	NOTE: A specific example for a drug library is the Oncology Set III from National Cancer Institute (NCI) Developmental Therapeutics Program (DTP). Compounds in this set are provided as 20 &#181;L at 10 mM in 100% DMSO in two 96-well PP U-bottom plates and stored at -20 &#176;C. The NCI Plate maps, and the basic chemical data of each drug could be found at the following websites: 
	<a href="https://dtp.cancer.gov/dtpstandard/servlet/PlateMap?searchlist=4740&#38;outputformat=html&#38;searchtype=plate&#38;Submit=Submit" target="_blank">NCI plate number 4740</a> 
	<a href="https://dtp.cancer.gov/dtpstandard/servlet/PlateMap?searchlist=4741&#38;outputformat=html&#38;searchtype=plate&#38;Submit=Submit" target="_blank">NCI plate number 4741</a></li>
</ol>
2. Screen for heterochromatin-promoting drugs using Drosophila
<ol>
	<li>Drosophila Breeding strategy (Figure 1A)
	<ol>
		<li>Day 1: Cross three w1118/Y; DX1/CyO male flies and three or more virgin w1118 female flies in a 25 mm x 95 mm food vial with 9 mL of standard Drosophila food media (Bloomington recipe). Set up three replicate vials for each drug and one control for the screen. 
		NOTE: DX1 flies were kindly provided by James Birchler (University of Missouri)9,13.</li>
		<li>Day 2 and Day 3: Allow the flies to lay eggs for 2 days at room temperature (22 &#176;C).</li>
		<li>Day 4: Use a short burst of CO2 gas to anesthetize the parent flies and dispose of the parent flies in a &#34;fly morgue&#34; (a bottle containing 70% alcohol).</li>
	</ol>
	</li>
	<li>Feed drugs to Drosophila for screening.
	<ol>
		<li>Dilute each drug compound from the original stock (20 &#181;L at 10 mM in 100% DMSO in 96-well PP U-bottom plates) to a 10 &#181;M final concentration with 33% DMSO in water. Use 33% DMSO in water without any drug as the control. 
		NOTE: Some drugs are poorly soluble in water and were dissolved in DMSO. 100% DMSO is toxic to flies. 33% is tolerable.</li>
		<li>Day 4: Pipette 60 &#181;L of a 10 &#181;M drug solution or control onto the top of fly food. At this point, ensure that the parent flies have been removed and there are fly eggs and crawling first-instar larvae on the food.</li>
		<li>Day 6: Repeat pipetting 60 &#181;L of the same 10 &#181;M drug solution to the food vial. 
		NOTE: Since drugs were added on to the fly food, the top surface food contains nearly 10 &#181;M concentration and the bottom food could be not be fully penetrated. All the flies lay eggs on the food top and eat the top surface food.</li>
	</ol>
	</li>
	<li>Observe and score eye color changes.
	<ol>
		<li>Two days after the F1 flies emerge (approximately on day 14), examine the flies. Use a short burst of CO2 to anesthetize the F1 flies and remove the flies from their vial onto a porous dissecting pad with CO2 sipping through from underneath a filter paper.</li>
		<li>Inspect the flies on this pad under a dissection microscope. Examine the whole fly and identify all w1118/Y; DX1/+ heterozygous males by their lacking the CyO dominant curly wing phenotype.</li>
		<li>Score the eye color of each of the w1118/Y; DX1/+ heterozygous male flies on a scale of 1 to 5 (Figure 1B). The percentage of white eye color was rated as follows: 
		1. &#60;5% red scattered eye total surface area 
		2. 6% - 25% red spots eye total surface area 
		3. 26% - 50% red spots eye total surface area 
		4. 51% - 75% red spots eye total surface area 
		5. &#62;75% red spots eye total surface area 
		NOTE: Alternatively, homogenize fly heads in 100% methanol and use a spectrometer to measure eye pigmentation at OD 450 nm. Select only males because PEV is more pronouced in DX1 males. Score more than 10 males in each vial.</li>
		<li>Calculate the mean color index of all the males from each vial scored. Perform triplicates for each compound (Figure 1C). 
		NOTE: Since eye color varies with age, this is a time-sensitive step. The eye color should be calculated at the same age &#8211; 2 days old. Usually &#62;10 flies should be scored in each vial.</li>
		<li>To confirm that the compounds indeed promote heterochromatin formation, use a different technique such as western blotting to validate (Figure 1G).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Morgan, M. A., Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+signatures+of+cancer.">Chromatin signatures of cancer.</a> Genes Development. 29 (3), 238-249 (2015).</li><li>Saksouk, N., Simboeck, E., Dejardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+heterochromatin+formation+and+transcription+in+mammals.">Constitutive heterochromatin formation and transcription in mammals.</a> Epigenetics Chromatin. 8, 3 (2015).</li><li>Allshire, R. C., Madhani, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+principles+of+heterochromatin+formation+and+function.">Ten principles of heterochromatin formation and function.</a> Nature Reviews Molecular Cell Biology. 19 (4), 229-244 (2018).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293 (5532), 1074-1080 (2001).</li><li>Grewal, S. I., Moazed, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+and+epigenetic+control+of+gene+expression.">Heterochromatin and epigenetic control of gene expression.</a> Science. 301 (5634), 798-802 (2003).</li><li>Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delving+into+the+diversity+of+facultative+heterochromatin:+the+epigenetics+of+the+inactive+X+chromosome.">Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome.</a> Current Opinion in Genetics &amp; Development. 15 (5), 482-489 (2005).</li><li>Ci, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+Protein+1alpha+Mediates+Development+and+Aggressiveness+of+Neuroendocrine+Prostate+Cancer.">Heterochromatin Protein 1alpha Mediates Development and Aggressiveness of Neuroendocrine Prostate Cancer.</a> Cancer Research. 78 (10), 2691-2704 (2018).</li><li>Zhu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-Encoded+Satellite+RNAs+Induce+Breast+Cancer.">Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.</a> Molecular Cell. 70 (5), 842-853 (2018).</li><li>Dorer, D. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+methotrexate+as+a+heterochromatin-promoting+drug.">Identification of methotrexate as a heterochromatin-promoting drug.</a> Scientific Reports. 9 (1), 11673 (2019).</li><li>Dialynas, G. K., Vitalini, M. W., Wallrath, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+Heterochromatin+Protein+1+(HP1)+to+cancer+progression.">Linking Heterochromatin Protein 1 (HP1) to cancer progression.</a> Mutation Research. 647 (1-2), 13-20 (2008).</li><li>Janssen, A., Colmenares, S. U., Karpen, G. 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The authors have no conflicts of interest to disclose.

Heterochromatin is a condensed form of DNA that plays a central role in regulating gene expression. It is becoming increasingly more recognized in cancer and may serve as a potential target for cancer therapy15,16,17,18. Small-molecule compounds are commonly used in drug development due to advantages in manufacture, preservation, and metabolism in human bodies. To identify heterochromatin-promo...

Heterochromatin is a condensed form of DNA that plays a central role in gene expression, in regulating chromosome segregation during cell division, and in protecting against genome instability1. Heterochromatin has been considered to be a gene repression regulator and to protect chromosome integrity during cell mitosis2,3. It is associated with the di- and tri-methylation of histone H3 lysine 9 (H3K9me) during lineage commitment4,5. Moreover, recruitment of Heterochromatin Protein 1 (HP1) chromodomain proteins is also considered to be associated with heterochromatin and epigenetic repression of gene expression6. These proteins are essential components and markers of heterochromatin formation.
Since genomic instability enables cells to acquire genetic alterations that promote carcinogenesis, heterochromatin is becoming more recognized in cancer development and may be targeted for cancer treatment7,8. Currently, there are no drugs that are well-established in assisting heterochromatin formation. Here, we present a simple and quick yet efficient method for screening small-molecule compounds that promote heterochromatin formation. The screening is done by treating Drosophila with a library of small-molecule drugs. This method takes advantage of a variegated eye color phenotype in the DX1Drosophila strain that is influenced by heterochromatin levels. DX1 flies contain a tandem array of seven P[lac-w] transgenes, which have the variegated expression/depression depending on heterochromatinization, therefore, the extent of variegation in the eye color reflect the heterochromatin level. Specifically, increasing heterochromatin could be detected by the rising proportion of variegated eye color (white eye). On the contrary, decreasing heterochromatin would be detected by the rising proportion of the P[lac-w] transgene expression (red eye)9,10,11,12.
Therefore, we take advantage of this Drosophila transgene system that produces a variegated eye color phenotype since its expression is directly correlated to the amount of heterochromatin present. Upon discovery of compounds that are suspected to promote heterochromatin formation, we may confirm this suspicion using other methods such as western blot. These heterochromatin-promoting substances may be further developed for clinical trials in patients in the future.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drosophila</td>
			<td></td>
			<td>DX1 strain</td>
			<td>DX1 flies were kindly provided by James Birchler (University of Missouri)</td>
		</tr>
		<tr>
			<td>Drosophila food media</td>
			<td>UCSD fly kitchen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methotrexate</td>
			<td>NCI drug library</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Sigma</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

a screening method for identification of heterochromatin-promoting drugs using drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila&#39;s DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.

This protocol was successfully used to screen compounds that promote heterochromatin formation in Drosophila, which is an efficient and low-cost in vivo system for drug development (Figure 1). We screened a small-molecule drug library, Oncology Set III, which is composed of 97 FDA authorized oncology drugs, using the DX1 strain of Drosophila melanogaster (Figure 1B). The results for a significant drug were represented ...

Watch this Scientific Journal Video about A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila at JoVE.com

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

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5.9K Views.  University of California San Diego. Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

Video: A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

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 ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Screening Cotton Genotypes for Reniform Nematode Resistance

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Here, a protocol is presented for the rapid non-destructive screening of cotton genotypes for reniform nematode resistance. The protocol involves visually examining the roots of nematode-infected cotton seedlings to determine infection response. The vegetative shoot from each plant is then propagated to recover plants for seed production.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:&#945;-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as &#8216;CID&#8217; gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...