This work was supported by a grant from the NHLBI to RW.

1. Power Tower Setup and Operation
<ol>
 <li>The Power Tower is placed on a rectangular piece of plywood that is clamped to a table. A 4.5 rpm gear motor from Grainger rests on top of four 2"x6" boards and one &frac34; inch plywood board. The motor, an AC/DC speed control, a fuse, and an on/off switch are wired together into a juncture box.</li>
 <li>To cool the motor, a hole 3 inches in diameter is cut into a vertical &frac12; inch piece of plywood. On one side, a 3-inch fan is attached to the plywood, and on the other, a 3-inch diameter metal cylinder is attached.</li>
 <li>A custom-made rotating arm with a roller mounted on the end is connected to the motor. The motor and attached arm rotate in a clockwise direction, pushing down one side of a bent &frac34; inch square tube stock that is attached to the plywood with a door hinge. A wheel is attached to the top side of the tube stock below the platform to allow it to roll against the platform that is directly above it.</li>
 <li>The arm pushes down on one end of the tube stock and lifts the opposite end, raising a two-level platform (Figure 1D). When the roller on the arm clears the tube stock it allows the arm to return to the starting position, dropping the platform back down (Figure 1F).</li>
 <li>The platforms consist of racks that are placed on top of plywood and secured by bungee cords that are hooked onto screw eyes. The platforms slide up and down along drawer sliders that are attached to the plywood with brackets.</li>
 <li>Vials are placed into the racks and secured by a square grid screen that is held in place with bungee cords.</li>
 <li>A Styrofoam cushion is placed below the platform to dampen the shock of the drop.</li>
 <li>The power of the motor is adjusted such that a full rotational period is 15 seconds long.</li>
</ol>
2. Exercise Protocol
<ol>
 <li>Flies are housed in a 25 &deg;C incubator with 50% humidity and a 12-hour light/dark cycle. Both exercise training and assessment of exercise capacity are conducted in a 25 &deg;C temperature-controlled room.</li>
 <li>Flies are collected and age-matched within 1-2 days of each other. A minimum of 240 flies is required for longitudinal monitoring of endurance and negative geotaxis (climbing) ability, two physiological indicators that reflect the effects of exercise training. Longitudinal monitoring allows isolation of induced change relative to starting values for each cohort, without confounding effects from variation between cohorts. Flies for additional experiments should be collected as needed.</li>
 <li>After collection, flies are divided into 120 experimental flies, which will be subjected to the exercise regimen, and 120 control flies, which will not be exercised. The flies are stored in vials of 20 flies each. The vials of experimental flies are plugged with firm cellulose acetate Flugs and the vials of control (unexercised) flies are plugged with soft sponge stoppers, which can be pushed down to immobilize the flies while on the Power Tower. Sponge is used for immobilization because its adaptive size allows easy placement near the bottom of the vial. The softer sponge material also reduces injuries caused by the more solid Flugs.</li>
 <li>Flies on the Power Tower are stored and exercised in vials that contain 5 mL of food, which provides them with a softer landing than flies that are exercised in empty vials. The food is typically composed of 10% sucrose, 10% yeast, and 2% agar in water, although we do not observe flies to eat often during the course of a training session. If empty vials are used during training, the percentage of flies that suffer injuries is dramatically increased.</li>
 <li>The Power Tower is operated in a temperature-controlled room that is maintained at 25 &deg;C. Experimental flies are placed on the Power Tower and made to climb. Control flies are also placed on the Power Tower, but the sponge stopper is pushed down into the vial approximately 3 mm above the flies in order to limit movement. This group serves as a control for any effects of the Power Tower that are unrelated to exercise. Although some room still exists for these flies to maneuver, we have repeatedly observed that flies under these conditions run very little and do not demonstrate a physiological response to exercise. It may also be desirable to include an additional control group that is not placed on the Power Tower at all.</li>
 <li>Flies are exercised five consecutive days each week, utilizing a ramped schedule (Figure 2). During week one the flies are exercised for 2 hours per session, week two for 2.5 hours per session, and week three for 3 hours per session. Although other regimens have been tested, this protocol produces consistent results across genotypes and under divergent conditions. This protocol is, however, subject to a wide array of possible variations that might suit a particular experiment.</li>
</ol>
3. Exercise and Locomotor Fatigue
<ol>
 <li>Time to fatigue during endurance exercise is a useful physiological indicator to confirm and/or quantitate exercise capacity of a given cohort. Fatigue assays can be performed longitudinally on cohorts that will be used in training experiments. Assays may be conducted either prior to training, after the completion of the training protocol, or both. Other intermediate time points can be added if charting incremental progress during training is desired. The fatigue assay can also be used to measure exercise endurance as a response to treatments other than the training protocol, such as diet alteration (Figure 3B).</li>
 <li>Fatigue assays should be conducted on a day when training is not taking place. Both the experimental and control flies are placed on the Power Tower in vials of 20 each and made to climb until fatigued. Fatigue is recognized as the failure to respond to a negative geotaxis stimulus with climbing behavior. Fatigue behavior is scored by visual observation.</li>
 <li>A vial of flies is considered "fatigued" when 5 or fewer flies are able to climb higher than 2 inches for four consecutive drops. Fatigued vials are removed from the Power Tower while it is still operating, and the time of removal is recorded. Times of removal can be plotted as a nested histogram, or as a "time-to-failure" plot. An example of data plotted as a "time-to-failure" plot is shown in Figure 3B.</li>
 <li>Vials are monitored at 10 minute intervals for a maximum of 10 hours (Figure 3B).</li>
</ol>
4. Exercise, Age, and Locomotor Ability
<ol>
 <li>Since this training protocol relies on induced running behavior, a primary assay for quantitating effects of training is to longitudinally measure change in average running speed during training. During the course of training, 120-fly cohorts of exercised and unexercised flies are tested daily for their response to a negative geotaxis stimulus. The RING technique described in Gargano et al. (2005) is used to measure the flies' climbing ability 12. Briefly, this technique measures average height climbed by a vial of flies during a defined time after induction of negative geotaxis. The height climbed during a defined time is equivalent to climbing speed. Measurements are performed before placing flies on the machine for daily training so as to avoid complications from fatigue after the day's run.</li>
 <li>In our standard protocol, flies are exercised for a total of three weeks and climbing ability is examined for a total of five weeks: three weeks during exercise-training and two weeks after cessation of exercise (Figure 2). This allows the experimenter to chart whether effects of exercise on mobility persist after cessation of the program.</li>
 <li>Four photographs of each cohort's daily RING performance are used for the analysis of climbing ability. Six vials are included in each photograph. Negative geotaxis is induced, and using a timed camera, a photograph is taken after a defined number of seconds. We typically use two seconds, although this can be varied depending on experimental conditions. For every photograph, each vial is divided into four quadrants of equal height, and image processing software is used to assign each fly a score based on the quadrant it reached within the allotted time period. Flies that climb to the topmost quadrant receive a score of 4, flies in the next highest quadrant receive a 3, flies in the second highest quadrant receive a 2, and flies in the lowest quadrant receive a 1. Flies that do not climb off of the bottom at all are given a score of 0.</li>
 <li>For each vial in a given photograph, a "climbing index" is generated by averaging the scores of all the flies in that vial. Each vial's indices from the four photographs are then averaged to give a final climbing index for that day.</li>
 <li>All negative geotaxis experiments are conducted longitudinally. Results are normalized to the initial "pre-exercise" score in order to reduce the effect of cohort variation in climbing ability and highlight the relative longitudinal change in a cohort's climbing capacity as the result of exercise training. The initial score is determined by the average of the first three days' climbing indices and is assigned a value of 1. Subsequent trials are then expressed as a percentage of the initial index.</li>
</ol>
5. Representative Results
Wild-type flies respond to an endurance protocol with a diminished age-related decrease in climbing ability that persists following the end of training, as reflected in longitudinal RING assays across five weeks of age (Figure 3A). This delayed decline in negative geotaxis is a standard phenotypic response that can serve as a positive control to ensure that wild-type exercise response is occurring normally. This data set is presented as an example of how the induction of exercise by the Power Tower program can be used as a behavioral input. The ability of various genetic or environmental factors to modulate this effect can then be assessed.
Conversely, the Power Tower can also be used as an output in various experimental designs. For example, genotype, diet, or other conditions can be varied. Then, the effect of these variations on exercise physiology can be tested using the Power Tower. Here, we show an example of this approach. When flies with a varying percentage of sucrose in their diets were tested for time to fatigue, increased sucrose content correlated with increased endurance capacity (Figure 3B).
<img alt="Figure 1" src="/files/ftp_upload/3786/3786fig1.jpg" /> 
Figure 1. Operation of the Power Tower. (A-C) The motorized bent arm with attached roller rotates clockwise until it comes into contact with the bent square tube. (D,E) The arm pushes down on the bent square tube, causing the platform that is laden with vials of flies to lift. (F) As the arm clears the tube the platform is allowed to fall back down, forcing the flies to return to the bottom of the vial.
<img alt="Figure 2" src="/files/ftp_upload/3786/3786fig2.jpg" /> 
Figure 2. Suggested Exercise Protocol. Flies subjected to the training protocol are made to exercise for five days each week under a three-week long ramped regimen that progressively increases the duration of exercise from the initial 2 hours by 30 minutes each week. Standard analyses include fatigue assays before and following the exercise program and RING assays from week 1 through week 5. All assays are performed in duplicate on an equal number of unexercised flies as a negative control. Other various physiological or biochemical tests can be conducted as determined by the researcher.
<img alt="Figure 3" src="/files/ftp_upload/3786/3786fig3.jpg" /> 
Figure 3. Endurance exercise alters multiple aspects of mobility. (A) RING assays performed longitudinally across ages in a single pair of male Y1W67C23 cohorts. Age-matched, genetically identical exercised and unexercised control flies were measured daily for average climbing speed. Results are expressed in terms of a climbing index that is normalized to the average climbing height across the first three days of measurement. Exercise-trained flies displayed a diminished age-related decline in negative geotaxis ability compared to age-matched unexercised siblings across ages (2-way ANOVA, p &lt; 0.005). (B) Fatigue assays conducted for 8 hours on age-matched female Canton S flies show a significant effect of dietary sucrose content on time to fatigue (Log-rank, p &lt; 0.0001). Prior to experiment, flies were fed a yeast/sucrose/agar diet, with 10% weight/volume yeast concentration, and a varying percentage of dietary sucrose. Five vials of 20 flies each were tested for each diet. Graph displays how many vials still have five or more flies running at a given time point. These results can be treated statistically and graphically as a survival (or time to failure) curve, with "failure" for a vial being defined as a point in time when less than five flies continue to respond to negative geotaxis stimulus. Note that many other possible study designs and statistical treatments are possible, and data treatment and measurement should be tailored to fit individual purposes.

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We have nothing to disclose.

The general protocol presented here has been successful in documenting physiological effects following training. However, several areas in this protocol are subject to modification to fit particular experimental needs. For example, the length of training and number of bouts could potentially be varied to make the program more or less challenging, as desired. The height of the container in which negative geotaxis ability is measured could be altered to increase the available area for improvement to be documented. Various methods of automating quantitation of climbing speed may also be applicable. In principle, any software program capable of distinguishing a fly from background can be used to speed the data gathering process.
Some aspects of the protocol should be modified only with great caution, however. For example, preliminary experiments strongly indicate that at least one day of rest per week tends to facilitate greater improvement than relentless daily exercise. Additionally, circadian rhythms and temperature are known to affect the movement of cold-blooded animals. The time of day that training takes place can be varied, but should always be consistent within particular groups under comparison, in order to avoid the possibility of confounding effects of circadian rhythms. Temperature control is also essential, and we recommend a dedicated room at constant temperature to house exercise equipment. Lastly, males and females must be reared and measured separately, in order to avoid the potential of confounding effects of fertility and sex differences in exercise capacity.
Potential applications of this methodology are limited only by the imagination of the researcher. In preliminary work, we have utilized this methodology in three broad applications:
<ol>
	<li>To measure the effect of endurance exercise on various aspects of wild type biology across ages.</li>
 <li>To measure the effect of endurance exercise on specific mutant phenotypes.</li>
 <li>To screen for genetic factors that are necessary to execute the benefits of exercise.</li>
</ol>
Each of these applications encompasses a wide variety of specific possibilities. Based on our preliminary experience, mutant phenotypes tend to vary with exercise level as much as they vary with diet. The use of invertebrate models to better understand the relationship between diet, exercise, and aging physiology is perhaps the most important general application of this protocol.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Dayton Gearmotor</td>
 <td>Grainger</td>
 <td>1LRA6A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Raco Electrical Box</td>
 <td>Grainger</td>
 <td>5A052</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Raco Cover</td>
 <td>Grainger</td>
 <td>5A053</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cooper Bussmann Fuse</td>
 <td>Grainger</td>
 <td>6F043</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cooper Bussmann Fuse Holder</td>
 <td>Grainger</td>
 <td>1DD33</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Carling Technologies Switch</td>
 <td>Grainger</td>
 <td>2X464</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dayton Control, AC/DC Speed</td>
 <td>Grainger</td>
 <td>4X796</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flugs for Narrow Plastic Vials</td>
 <td>Genesee Scientific</td>
 <td>49-102</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

endurance training protocol and longitudinal performance assays for drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Watch this Scientific Journal Video about Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster at JoVE.com

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

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15.5K Views.  University of Michigan Medical School. We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Video: Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.