We thank George Watase and Josie Clowney for discussions during the initial stages of the protocol development. The work was supported by funds from the NIH (grant no. R35GM133737 to S.Y.), Alfred P. Sloan Fellowship (to S. Y.) and McKnight Scholar Award (to S. Y.).

1. Design and construction of gene editing reagents
NOTE: To carry out endogenous tagging, it is essential to identify the various isoforms of the desired gene. Depending on the specific goals of the experiment, a fluorescent tag can be incorporated either internally or at the N- or C-termini of the selected protein isoform(s). For optimal CRISPR-mediated editing, it&#39;s crucial to position the two sgRNAs suitably close to the intended knock-in site. Subsequently, to enable editing through hom...

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+strategies+for+protein+trapping+in+Drosophila.">Exploring strategies for protein trapping in Drosophila.</a> Genetics. 175 (3), 1089-1104 (2007).</li><li>Sarov, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+resource+for+the+analysis+of+protein+localisation+in+Drosophila.">A genome-wide resource for the analysis of protein localisation in Drosophila.</a> Elife. 5, e12068 (2016).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8, 2281-2308 (2013).</li><li>Port, F., Chen, H. M., Lee, T., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+CRISPR/Cas+tools+for+efficient+germline+and+somatic+genome+engineering+in+Drosophila.">Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.</a> Proc Natl Acad Sci U S A. 111 (29), E2967-E2976 (2014).</li><li>Gratz, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+specific+and+efficient+CRISPR/Cas9-catalyzed+homology-directed+repair+in+Drosophila.">Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.</a> Genetics. 196 (4), 961-971 (2014).</li><li>Kanca, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+CRISPR-based+strategy+to+insert+small+and+large+fragments+of+DNA+using+short+homology+arms.">An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.</a> Elife. 8, e51539 (2019).</li><li>Lamb, A. M., Walker, E. A., Wittkopp, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+strategies+for+scarless+allele+replacement+in+Drosophila+using+CRISPR/Cas9.">Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.</a> Fly (Austin). 11 (1), 53-64 (2017).</li><li>Xiao, Y., Yuan, Y., Jimenez, M., Soni, N., Yadlapalli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clock+proteins+regulate+spatiotemporal+organization+of+clock+genes+to+control+circadian+rhythms.">Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms.</a> Proc Natl Acad Sci U S A. 118 (28), e2019756118 (2021).</li><li>Costantini, L. M., Fossati, M., Francolini, M., Snapp, E. 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The results demonstrate a streamlined, simple one-step CRISPR-based strategy for tagging endogenous genes in Drosophila. In the protocol, we use two gRNAs to induce DNA double-strand break and homologous recombination more efficiently. The gRNAs and the donor plasmid are injected into fruit fly embryos, which express Cas9 enzyme in their germline. These embryos are subsequently cultivated under standard conditions until adulthood, at which point they are crossed with balancer flies to produce the first generatio...

Fluorescent proteins have emerged as powerful tools for visualizing protein localization, dynamics, and protein-protein interactions in cells within living organisms1,2. Tagging endogenous genes with fluorescent proteins enables long-term live imaging of cellular processes with subcellular resolution. Furthermore, these endogenous gene-labeling techniques have several advantages compared to overexpression and immunostaining approaches. For example, overexpression of proteins might lead to protein misfolding, nonspecific protein-protein interactions, and mislocalization3. To date, researchers have been able to create vast libraries of endogenous genes fused to fluorescent proteins for a few model organisms, such as budding yeast, which can undergo efficient homologous recombination4. In the case of Drosophila melanogaster, various tools such as MiMICs5, transposon-based enhancer traps6, and fosmids7 have been devised to fluorescently tag genes.
The development of CRISPR-Cas9-based tools has made it possible to efficiently perform genome editing, including tagging endogenous proteins, in a wide range of model organism8,9. Cas9 is an RNA-guided enzyme that cleaves double-stranded DNA in a highly efficient and specific manner8, which can then be repaired by a nonhomologous end-joining (NHEJ) pathway leading to point mutations or deletions. Alternatively, the homology-directed repair (HDR) pathway allows the integration of heterologous DNA into the chromosome.
Past methods for CRISPR-based gene tagging in the model organism, Drosophila melanogaster, have relied on a two-step process10,11,12. This involves using a discernible eye-color marker, Dsred, to detect successful HDR events. Afterward, the Dsred marker would be excised using the Cre/loxP recombination system. To streamline and expedite this process, here, we present a simpler and more efficient method. This approach circumvents the need for lethal genotyping and allows for the direct screening of individual flies. Specifically, we employ a PCR-based approach to extract DNA from a small segment of the fly&#39;s middle leg, allowing us to genotype individual flies. Notably, this procedure does not compromise the vitality, movement, or reproductive capabilities of the sampled fly. Only the appropriate individuals, identified through this method, are further developed into stable stocks.
Here, we present a detailed protocol for generating fluorescent protein tagged flies in which endogenous genes are labeled with fluorescent reporters. This technique uses CRISPR-Cas9 genome editing to fuse any desired fluorophore tag to the N- or C- terminal of an endogenous gene. Below, we describe the design and construction of gRNA plasmids and a donor plasmid, one-step screening strategy to select CRISPR-positive flies, and steps to establish stable lines. Specifically, we describe strategies to tag an endogenous core clock Drosophila gene, period, with a fluorophore at the C-terminal. We also briefly describe the control experiments that need to be performed to confirm that the tagged protein is functional and live-imaging techniques to visualize the Period protein in live clock neurons within intact Drosophila brains13. The protocol described here is also broadly applicable to screen candidates for deletions and insertions of specific bits of DNA by CRISPR-Cas9 genome editing.

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			<td>0.5M EDTA pH8.0</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>5-alpha Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td>NEB</td>
			<td>C2987H</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well deep well plate</td>
			<td>BRAND</td>
			<td>701354</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BbsI-HF</td>
			<td>NEB</td>
			<td>R3539S</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
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			<td>D-Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
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		<tr>
			<td>EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101S</td>
			<td></td>
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			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A142-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Glass Antifade Mounting Medium</td>
			<td>Invitrogen</td>
			<td>P36982</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>QIAGEN</td>
			<td>19131</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila Medium</td>
			<td>Gibco</td>
			<td>21720001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td></td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>NEB</td>
			<td>R0145S</td>
			<td></td>
		</tr>
	</tbody>
</table>

one-step crispr-based strategy for endogenous gene tagging in drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

In our experiments, we typically found a success rate of ~20%-30% in screening for various endogenous tagged genes on all chromosomes (X, II, III). The efficiency of this process can vary based on several factors, such as gRNA selection, the nature of the gene, and injection quality. Here, we illustrate the results from our strategy to tag the endogenous period gene with the mNeonGreen fluorophore13. The period gene, positioned on the X chromosome...

Watch this Scientific Journal Video about One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster at JoVE.com

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

one-step-crispr-based-strategy-for-endogenous-gene-tagging-drosophila

Research

JoVE Journal

Neuroscience

455 ビュー.  University of Michigan. このプロトコルの目的は、内因性編集されたフライラインを生成することです。ここでは蛍光タンパク質のノックインを例に挙げていますが、他のノックイン、ノックアウト、突然変異の生成にも応用できます。私たちのプロトコルはCRISPR相同組換えベースの方法であるため、変異部位に制限はほとんどありません。さらに、当社のプロトコルは、ジェノタイピン?...

ビデオ: One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in...

endogenous protein tagging in human induced pluripotent stem cells using crispr/cas9

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are...

visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

all-in-one crispr genome editing: a method for homology directed repair-based gene knock-in in cultured cells using crispr-cas9 system

In this video, we demonstrate all-in-one CRISPR-Cas9 based genome editing in cultured cells where Cas9 and sgRNA are provided as a single plasmid construct to the cells. The CRISPR-Cas9 system and desired gene to be inserted was introduced in cells through electroporation technique to facilitate successful gene...

All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System

ubiquitous and tissue-specific rna targeting in drosophila melanogaster using crispr/casrx

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx

This article outlines a detailed protocol for using the RNA-targeting Cas13D enzyme (RfxCas13D) in flies.

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx

Prepare cell-DNA mixture for electroporation. Collect JURKAT cells culture in 25-square-centimeter flask. Transfer cells into 50-mL centrifuge tube. Then, pellet the cells by centrifugation at 90 x g for eight minutes at room...

versatile co2 transformations into complex products: a one-pot two-step strategy

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations are conducted in a one-pot two-step procedure for the synthesis of complex molecules. The selective 4 e- reduction of CO2 with a hydroborane reductant affords a reactive and versatile bis(boryl)acetal intermediate which is subsequently involved in condensation reaction or carbene-mediated C-C coupling...

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

preparation of the mgm101 recombination protein by mbp-based tagging strategy

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion...

a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

crispr interference-based gene silencing: a technique for targeted repression of gene function in pathogenic leptospira

This video demonstrates a CRISPR interference-based gene silencing in pathogenic Leptospira. Targeted repression of gene expression using a CRISPR/Cas system that blocks mRNA formation helps identify candidate genes for pathological and pharmacological...

CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira

high-throughput gene tagging in trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is...

High-throughput Gene Tagging in Trypanosoma brucei

gene digital circuits based on crispr-cas systems and anti-crispr proteins

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

an efficient strategy for generating tissue-specific binary transcription systems in drosophila by genome editing

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

measurement of lifespan in drosophila melanogaster

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...

Measurement of Lifespan in Drosophila melanogaster

Next, in the day of conjugation, grow the saturated E. coli cultures in LB plus DAP. Inside a biosafety hood, assemble the filtration apparatus by placing the membrane filter on top of the glass base, followed by 15-mL glass funnel. Both pieces are held together by the spring clamp. Then, connect the glass to a vacuum pump. Add 5 ml of Leptospira cultures to the...

monitoring heart function in larval drosophila melanogaster for physiological studies

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...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

assay for blood-brain barrier integrity in drosophila melanogaster

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

quantifying abdominal pigmentation in drosophila melanogaster

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.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...