Name: using the gal4-uas system for functional genetics in anopheles gambiae
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We gratefully acknowledge funding from LSTM and IVCC (Adriana Adolfi), BBSRC (New Investigator Award (AL), MRC (PhD studentship to BCP:MR/P016197/1), Wellcome (Sir Henry Wellcome Postdoctoral fellowship to LG: 215894/Z/19/Z) that have incorporated Gal4UAS analysis in the proposals.

1.&#160;Design and construction of UAS constructs
<ol>
	<li>Design and assembly of vectors for candidate gene expression
	<ol>
		<li>Determine the sequence to be used for candidate gene upregulation.
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			<li>Sequence the cDNA/gDNA from the strain of interest and compare it to the published sequence to verify its identity and identify potential SNPs and restriction sites for diagnostic digest.</li>
			<li>Ensure that the forward primer used for gene amplification covers the native...

<ol>
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Understanding mosquito gene function is vital to develop new approaches to control&#160;Anopheles&#160;and impact malaria transmission. The GAL4-UAS system described is a versatile and powerful system for functional analysis of candidate genes and to date we have used the system to examine the genetic basis of insecticide resistance17&#160;and cuticular hydrocarbon production15,23, as well as to fluorescently tag different mosquit...

The bipartite GAL4-UAS system is the workhorse of functional characterization of genes in the insect model organism Drosophila melanogaster1,2,3. To use the GAL4-UAS system, transgenic driver lines, expressing the yeast transcription factor GAL4 under control of a regulatory sequence, are crossed with responder lines carrying a gene of interest or RNA interference (RNAi) construct controlled by an Upstream Activation Sequence (UAS) recognized by GAL4. The progeny of this cross express the transgene of interest in a spatiotemporal pattern dictated by the promoter controlling GAL4 expression (Figure 1). Phenotypes displayed by progeny of driver-responder crosses can be assessed to elucidate the function of candidate genes. Although D. melanogaster has been used to examine genes from other organisms4,5,6,7, the GAL4-UAS system has now been adapted for use in insects of medical and agricultural importance to provide direct analysis in the species of interest 8,9,10,11,12,13,14.
In the African malaria mosquito, Anopheles&#160;gambiae, the GAL4-UAS system was first tested by cell line co-transfection9. Multiple constructs were assayed for efficiency in different pairwise combinations and found that 14 tandemly repeated UAS supplemented with a small artificial intron (UAS-14i) displayed the widest range of activation potential when used with a panel of GAL4 drivers. To demonstrate in vivo functionality, these constructs were then used to create two separate transgenic An. gambiae lines by PiggyBac transformation8: a driver line carrying GAL4 driven by a midgut specific promoter, and a responder line containing both the luciferase and enhanced yellow fluorescent protein (eYFP) genes under regulation of UAS sequences. Gut specific luciferase activity and fluorescence in the progeny indicated that the system was efficient in Anopheles. Since then, driver lines have been created expressing transgenes in other tissues important for vectorial capacity and insecticide resistance, including oenocytes15 and hemocytes16, and in a close to ubiquitous pattern10. Numerous UAS lines have also been generated to assay genes thought to be involved in metabolism and sequestration mediated insecticide resistance, cuticular hydrocarbon synthesis and to fluorescently tag different cell and tissue types (Table 1). For the responder lines, site-directed integration of the transgene is now performed by &#934;C31 catalyzed recombination cassette exchange17,18 to fix the genomic context of the UAS regulated genes. In this way, transgene expression is normalized regarding genomic insertion location, allowing for more accurate comparison of the phenotypic effects of different candidate genes.
The responder lines created to date are designed to either express the transgene either at elevated levels or to reduce gene expression through RNA interference (RNAi). Usually cDNA clones are fused to the UAS sequence to generate suitable expression plasmids, however full genomic sequences are also feasible assuming that they are not too large for cloning. To generate silencing constructs, we have used three different methods to obtain suitable tandem inverted sequences that form hairpin dsRNA that stimulates RNAi. These have included fusion PCR, asymmetric PCR and commercial synthesis of hairpin constructs. Common to each method is the inclusion of an intron sequence between the inverted&#160;sequences to provide cloning stability. Responder plasmids into which a gene of interest/RNAi construct can be inserted have been developed15. These plasmids also carry the required &#934;C31 attB sites for RMCE (described in Adolfi accompanying JoVE paper which describes the RCME technique in detail). Protocols covering the important steps required when selecting the sequence for insertion into one of these plasmids for overexpression are included in this manuscript. Additionally, two protocols for RNAi hairpin construct creation are described and illustrated.
When creating new lines, identification of rare transgenic individuals is crucial to breed from to establish and maintain transgenic colonies. Most importantly for the GAL4-UAS system there is a necessity to distinguish the responder and driver lines to establish crosses and identify individual progeny that carry both transgenes. This is achieved by using different dominant selectable marker genes linked to the driver and responder cassettes. Most commonly these are fluorescent marker genes that are clearly distinguishable using optical filters (e.g., eYFP, eCFP, dsRed). It is important that markers are expressed in a known and reliable spatiotemporal pattern as this makes identification of abnormalities and contamination easier. Fluorescent marker gene expression is routinely regulated by the synthetic 3xP3 promoter, which causes eye and ventral ganglia specific expression in all stages of An. gambiae development19. Fluorescent markers controlled by 3xP3 are included in all transformation plasmids described in this article. A protocol detailing the common methods used to screen fluorescent An. gambiae pupae GAL4-UAS lines is included here.
One of the key elements of the GAL4-UAS system is the necessity to cross the differentially marked driver and responder lines. To do this male and females from each line must be separated prior to mating. Adults are readily distinguishable by sight, however, for establishing genetic crosses it is sensible to separate the sexes prior to adult emergence to ensure that mating has not occurred. The general size difference between male and female An. gambiae pupae is too variable to be an efficient and dependable method of sex determination20. Instead clear morphological differences in the external genitalia provide a reliable basis for sexing in An. gambiae. In this article, we describe a dependable method for sexing An. gambiae pupae to set up appropriate crosses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62131/62131fig1v3.jpg" /> 
Figure 1&#160;- Diagrammatic representation of process for using the bipartite GAL4-UAS System in Anopheles gambiae.&#160;(A) The major components of an example vector (pSL-attB-UAS14-gyp[3xp3-eYFP]) are depicted, detailing the available restriction sites (EcoRI, NheI, XhoI and NcoI) within the multiple cloning sites that are suitable for use to insert the hairpin construct or coding sequence for the gene of interest. The structure of the docking line is also depicted. (B) The crossing step is illustrated indicating the use of males from the driver line (carrying GAL4 driver by a promoter of interest and eCFP driven by the 3xP3 promoter) and females from the responder line (carrying the gene of interest or hairpin construct controlled by a UAS promoter and an eYFP marker controlled by the 3xP3 promoter). (C) A diagrammatic representation of GAL4 driving expression of the gene of interest in the progeny of the cross in B and a list of some of the typical phenotypes that are assessed. Abbreviations: Multiple Cloning Site (MCS), Recombinase mediated cassette exchange (RMCE), Upstream Activator Sequence (UAS), enhanced yellow fluorescent protein (eYFP), enhanced cyan fluorescent protein (eCFP). <a href="https://www.jove.com/files/ftp_upload/62131/62131fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is the use of crosses that provides the bipartite nature of the GAL4-UAS system, which has distinct advantages over more linear approaches. For example, many more combinations of driver and responder lines can be assessed than would be feasible if a new transgenic line had to be generated and maintained for each promoter/gene combination. More importantly, it allows the analysis of genes that produce lethal or sterile phenotypes when their expression is perturbed which are difficult to create/maintain in a linear system. Such lethal phenotypes can manifest at all developmental stages, depending on the gene function and spatiotemporal expression, but are most often observed during embryonic development. Visualizing mosquito embryo development requires the clearing of the opaque chorion which coats the eggs. Following methods described in Trpi&#353; (1970)21 and Kaiser et al. (2014)22, we describe the protocols we use to fix embryos, whilst maintaining structural integrity, and bleaching to clear the endochorion that allows microscopic visualization and imaging.

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	<tbody>
		<tr>
			<td>100 x 15 mm plastic Petri dish</td>
			<td>SLS</td>
			<td>2175546</td>
			<td>Pack of 10</td>
		</tr>
		<tr>
			<td>1000 &#181;L Gilson Pipette</td>
			<td>Gilson</td>
			<td>F144059P</td>
			<td></td>
		</tr>
		<tr>
			<td>20/25 mL Universal Tubes</td>
			<td>Starlab</td>
			<td>E1412-3020</td>
			<td>Pack of 400</td>
		</tr>
		<tr>
			<td>3 mL Pasteur Pipettes</td>
			<td>SLS</td>
			<td>G612398</td>
			<td>Greiner Pasteur pipette 3 mL sterile individually wrapped</td>
		</tr>
		<tr>
			<td>50 mL Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>11512303</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818-500</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>SLS</td>
			<td>45726-1L-F</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Cages</td>
			<td>SLS</td>
			<td>E6099</td>
			<td>30x30x30 with screen port</td>
		</tr>
		<tr>
			<td>Fine Paint Brushes</td>
			<td>Amazon</td>
			<td>UKDPB66</td>
			<td>KOLAMOON 9 Pieces Detail Painting Brush Set Miniture Brushes for Watercolor, Acrylic Painting, Oil Painting (Wine Red)</td>
		</tr>
		<tr>
			<td>Fish food</td>
			<td>Amazon</td>
			<td></td>
			<td>Tetra Min Fish Food, Complete Food for All Tropical Fish for Health, Colour and Vitality, 10 L&#160;</td>
		</tr>
		<tr>
			<td>Formaldehyde Solution</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouth Aspirator</td>
			<td>John Hock</td>
			<td>612</td>
			<td></td>
		</tr>
		<tr>
			<td>Pond Salt</td>
			<td>Amazon</td>
			<td></td>
			<td>Blagdon Guardian Pond Tonic Salt, for Fish Health, Water Quality, General Tonic, pH Buffer, 9.08 kg, treats 9,092 L&#160;</td>
		</tr>
		<tr>
			<td>Pupae Pots</td>
			<td>Cater4you</td>
			<td>SP8OZ</td>
			<td>250 pots with lids</td>
		</tr>
		<tr>
			<td>Small Plastic Buckets</td>
			<td>Amazon</td>
			<td></td>
			<td>2.5 L White Plastic Pail Complete with White Lid (Pack of 10)&#160;</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>S25552</td>
			<td></td>
		</tr>
	</tbody>
</table>

using the gal4-uas system for functional genetics in anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa.
In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

3xP3 expression of eYFP, dsRed and eCFP provides reliable, readily distinguishable identification of individuals possessing the marker genes producing expression in eyes and ventral ganglia of&#160;An. gambiae&#160;pupae (Figure 3). The differential morphology observed in male and female external genitalia used for sexing and an example of an unidentifiable pupae are highlighted in&#160;Figure 4. Removal of all water from pupae increases sexing...

Watch this Scientific Journal Video about Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae at JoVE.com

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

The bipartite GAL4-UAS system is a versatile tool for functional genetic analysis in Anopheles gambiae through genetic modification of gene expression in a spatiotemporal controlled manner. The binary system enables flexibility in experimental approaches for phenotypic characterization of transgene expression even if severe fitness costs are induced. This system can be used for phenotypic characterization of genes potentially involved in insecticide resistance, pathogen transmission, or those that have uses in genetic control methods.Fraser Coleman, a research technician, will help to demonstrate this procedure Begin by collecting pupae using a three-milliliter plastic Pasteur pipet with the end cut off onto a clear flat dish suitable for use with a stereo microscope. Carefully remove most of the water around the pupae. Turn on the fluorescent bulb and allow it to warm up.Consider the color spatiotemporal patterns of a expression and ratio of different phenotypes that are expected when screening for fluorescent marker. Select the required filter on the fluorescence stereoscope and check that there is a colored beam of light visible that is directed at the center of the stage plate. Under white light, center the pupae in the field of view and bring them into focus.Turn on off the white light and use the fine focus to bring the area of the pupae carrying the phenotype of interest with a visible fluorescent pattern into focus. Use a fine detailing paintbrush to gently turn the pupae and move the anal paddles so that the external genitalia can be identified. Separate pupae based on distinctive external genitalia.Males have a long tube that extrudes from the final dorsal segment approximately half the length of the anal paddles. The external genitalia of female pupae are considerably shorter and bifurcate. Separate the sexed pupae into groups of 10 or less in clear 20-milliliter tubes with water, and seal the tubes with a ball of cotton wool.Aspirate the desired number of male and female adults from the tubes into a cage or small bucket, taking care not to damage the adults during this transfer. Maintain the cross for five to seven days before blood feeding. On the following day, eggs can be collected to study embryo development.Assemble the oviposition chamber and carefully introduce 10 to 15 blood-fed females into it. Cover the oviposition chamber and leave for 20 minutes. Carefully detach the 50-milliliter polypropylene tube from the oviposition pot, making sure not to release the mosquitoes.Cover the pot and allow eggs to mature to the developmental stage of interest. Pick up eggs from the pot using a fine detailing paintbrush and place them on water in a 40-square millimeter excavated glass block. Carefully remove the water from the glass block with a micropipette and cover eggs in 500 microliters of FAA.Oscillate gently on orbital shaker at room temperature for 30 minutes. Thoroughly rinse the eggs 15 times with distilled water to remove all traces of FAA solution. Using a 1000-microliter micropipette add and then remove one milliliter of distilled water at a time taking care not to damage the eggs while doing so.Cover the fixed eggs with one milliliter of Trpis solution and incubate at room temperature for 30 minutes. Eggs will start to develop pale patches after five minutes of incubation eventually reaching a milky white color once cleared. Rinse the eggs thoroughly 15 times with distilled water to remove all traces of Trpis solution.The expression of fluorescent markers driven by the 3xP3 promoter is observed in the eyes and ventral ganglia of Anopheles gambiae pupae. Using different colored fluorescent markers permits the selection and screening of modified individuals carrying different GAL4 and UAS components. When working with a bipartite GAL4-UAS system, it is necessary to cross males and females from different strains to acquire progeny carrying both the GAL4 and UAS components.The differential morphology observed in male and female external genital of pupae is used for sexing. An example of an unidentifiable pupae is highlighted here. Removal of all water from pupae, as is shown on the right, increases sexing difficulty as anal paddles obscure visualization of genitalia.The bipartite GAL4-UAS system permits modification of gene expression in a controlled spatiotemporal manner. This can be visualized by crossing the oenocyte and ubiquitous GAL4 four expressing driver lines with the responder line UAS-mCherry. A key benefit of using a bipartite GAL4-UAS system is the ease of studying lethal phenotypes even at the embryonic stage.To do this, it is necessary to visualize the internal morphology of the embryos. The normal morphological characteristics observed in different stages of embryonic development at 12, 24, and 36 hours post-laying can be seen following completion of the embryo clearing protocol. It is vital throughout this procedure that pupae and eggs are not allowed to desiccate.Therefore, it's important to work quickly when removing liquids. The GAL4-UAS method has allowed us to functionally characterize genes involved in insecticide resistance and those with potential uses in gene drive control methods.

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Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...