Name: measuring exercise levels in drosophila melanogaster using the rotating exercise quantification system (reqs)
Uploaded: 2018-05-27T15:00:00
Description: Watch this Scientific Journal Video about Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS at JoVE.com

The work was supported by Award Number P30DK056336 from the National Institute Of Diabetes And Digestive And Kidney Diseases through a pilot grant from the Nutrition and Obesity Research Center at the University of Alabama at Birmingham to NCR.

The REQS consists of a Drosophila Activity Monitor unit (for source information, please see Table of Materials) mounted on a rotating arm that is controlled by a motor unit (Figure 1). The activity monitor determines how often in a given time span the array of laser beams dissecting the middle of the assay tube is disrupted. For detailed drawings and an in-depth characterization, see our previous publication9. While our system uses the LAM25H unit, th...

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As the representative results illustrate, the REQS is capable of accurately measuring the activity of exercising Drosophila. The REQS is flexible and allows researchers to address a variety of research questions related to exercise biology or exercise interventions. There are two critical steps in the protocol to highlight. First, it is essential to test the setup of the REQS to ensure that data transmission from the REQS to the DAMSystem308 is working properly. If not properly set up, the data cable can become tangled d...

Recently, researchers have begun to use the fruit fly Drosophila melanogaster to study exercise biology. D. melanogaster has been a genetic model system for over 100 years1,2. However, Drosophila research has made contribution to not just genetics, but also to a variety of other disciplines including neurobiology, behavioral biology, and physiology3. In 2009, the Power Tower, the first exercise machine for Drosophila was described4. The Power Tower takes advantage of the animals&#39; negative geotaxis response. When disturbed, Drosophila tend to move to the top of their enclosure. This response is well established and is the basis of the popular &#34;RING&#34; (Rapid Iterative Negative Geotaxis5) assay that is used to estimate climbing ability and/or physical fitness in Drosophila. The Power Tower uses a mechanical arm connected to a motor unit to repeatedly lift a set of animals within their enclosures by several inches and dropping them back to the ground to induce the negative geotaxis response (Tinkerhess et al. 20126 provide a video illustrating the use of the Power Tower). Prolonged treatment on the Power Tower thus increases the amount of physical activity (running or flying) the animals perform compared to untreated control animals and over time leads to improved performance in the RING assay for physical fitness4. Thus, this work demonstrated the feasibility of using Drosophila as a model for exercise biology.
To expand the repertoire of tools available for Drosophila exercise research, in 2016, Mendez and colleagues described a second Drosophila exercise machine, the Treadwheel7. Similar to the Power Tower, the Treadwheel exploits the negative geotaxis response of Drosophila. However, this response is induced by continued rotation of the animal enclosures, rather than by lifting and dropping them as in the Power Tower. This induction method is gentler and allows for a more endurance oriented exercise regime that avoids any physical trauma that might occur during exercise in the Power Tower (see Katzenberger, R. J. et al. 20138 for the impact of repeated physical trauma on Drosophila health). Similar to the Power Tower4, exercise treatment of animals on the Treadwheel leads to a variety of physiological responses, including changes in physical fitness, triglyceride levels, and body weight7. Thus, two complementary methods are available for Drosophila biologists studying exercise.
One limitation of both the Power Tower and the Treadwheel is the inability to measure the amount of activity induced by the exercise treatment. Analysis of video-recordings taken from the Treadwheel demonstrated that there were significant differences among the various Drosophila strains in how they respond to the exercise treatment7. Specifically, the strains studied differed in how much additional activity the animals performed when stimulated7. This observation prompted us to develop a third exercise system, the Rotating Exercise Quantification System (REQS), that allows us to measure animal activity levels during rotation-induced exercise9. The REQS utilizes a commercially available activity monitoring unit that is installed on a rotating arm to stimulate exercise through rotation as in the Treadwheel. Initial work with the REQS confirms that genetically different Drosophila strains &#8212; and sexes - can have significantly different responses to the rotational stimulation and thus the amount of exercise induced is not identical among different genotypes9. Thus, the REQS now enables Drosophila biologists to measure the amount of exercise induced by the treatment, opening a variety of new research avenues in the exercise field.
Here we describe in detail how to use the REQS for quantification of rotational exercise. The REQS induces rotational exercise and simultaneously measures the activity levels of the animals being treated. The REQS is able to accommodate a variety of exercise programs, ranging from the simple 2 h continuous exercise regime demonstrated here to more complex interval training methods as described by Mendez and colleagues7, and stimulation can be adjusted via rotation speed (between approximately 1-13 rotations per min). Depending on the activity monitor unit used to produce the REQS, this method is adaptable to the analysis of single flies or large populations of animals. Due to this versatility, the REQS provides Drosophila researchers with an array of opportunities to study, for example, different exercise regimes, diet interventions, or impact of population density.

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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.

The output from an individual run with the REQS is a data table produced by the DAMSystem308 software, which will be labeled &#34;Monitor1.txt&#34; by default (for an example see Supplemental File 1). An excerpt from such a table is shown in Table 1. Each column contains the data from an individual assay tube, while the rows contain the activity measured in each time interval from the beginning of the experiment (top) to the end (bottom). The first three ...

Watch this Scientific Journal Video about Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS at JoVE.com

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.

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 ...

This method can help answer key questions in the field of exercise biology, such as how does exercise response differ for animals of different ages and what genetic pathways control exercise response. The main advantage of this technique is that it allows researchers, for the first time, to measure activity in flies performing rotationally-induced exercise in real time. Demonstrating the procedure will be Louis Patrick Watanabe, a graduate student from my laboratory.Prior to the planned experiment, set up crosses of male and female flies so that each controlled progeny can be collected for the experiment. To begin, load eight glass specimen tubes with 10 anesthetized test flies per vial. A standard experimental group consists of 10 vials of 10 flies of each sex.To load the vials into the REQS, insert the vial into a slot of the activity monitor from the front. Then, put the second cap on the vial and center the vial, moving the O-ring as needed. House the REQS in an incubator for controlled environmental conditions.Allow the animals to recover from the anesthesia for an hour in this incubator before proceeding. For assays longer than eight hours, vials containing food that is very securely attached to one end of the tube may be used. Following the acclimatization period, power up the device to start the rotations.Adjust the speed to the desired speed, such as four RPM. No tube should make contact with the bottom surface if they are properly positioned. For a typical assay, simply initiate the software and record the activity for the desired time, such as for two hours when using vials without food.Upon initiation, the software automatically saves the data to a text file. In the software, a green connection light indicates the software is collecting data. The recording frequency can be adjusted under the Preferences tab.Typically, five minute intervals are used. During the assay, do not open the incubator, as the animals are very sensitive to any disturbance and are likely to respond with increased activity. At the end of the assay, terminate the data collection by closing the software, then turn off the REQS and inspect the tubes.Take note of any deaths that occurred during the exercise regime. Generally, four RPM for two hours is not lethal to healthy strains. Finally, unload the vials by anesthetizing the flies with carbon dioxide.The data file can be analyzed with any statistical package. If there are time points where an activity score of zero is recorded for all positions, then there was a cut out in the connection during these time points. If cut outs occur, stabilize the connections leading to the rotating phone jack using tape.Four genetically distinct drosophila lines were assayed with the REQS using the described protocol. Each group was composed of 100 flies split into 10 vials. The average activity was calculated in five minute bins over the two hour assay, conducted at four RPM.The mean exercise activity between all four genotypes was significantly different, however, no significant difference was found between virgin male and virgin female flies, so their data is presented together in this analysis. After its development, this technique paved the way for researchers in the field of exercise genetics to explore various exercise responses in drosophila melanogaster and have studied the impact of exercise on animal behavior and physiology. Following this procedure, other methods, such as body composition analysis, stress tests, and fitness tests can be used to answer additional questions regarding the impacts of exercise treatments on the animals.

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Genetics

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

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 ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

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 ...

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.

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.

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' ...