We would like to thank Hugo Bellen and the Bloomington Stock Center for reagents. We further thank Koen Venken, Hugo Bellen and all members of the Buszczak and Hiesinger labs for helpful discussions. This work was supported by grants from the National Institute of Health to ACR (T32GM083831), PRH (RO1EY018884) and to MB (RO1GM086647), a grant by the Cancer Prevention Research Institute of Texas to MB and PRH (RP100516), and the Welch Foundation (I-1657) to PRH. MB is an E.E. and Greer Garson Fogelson Scholar in Biomedical Research and PRH is a Eugene McDermott Scholar in Biomedical Research at UT Southwestern Medical Center.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Venken, K. 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=Versatile+P[acman]+BAC+libraries+for+transgenesis+studies+in+Drosophila+melanogaster.">Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster.</a> Nat. Methods. 6, 431-434 (2009).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, M. 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The authors do not have any competing interests in regards to the techniques outlined here.

The power of genetic model organisms in biomedical research is largely based on the tools available for genetic manipulation. The small models C. elegans and Drosophila in particular allow for inexpensive and fast molecular genetic analyses of complete pathways and gene families implicated in multicellular development or function. Recent years have seen significant advances in tool development for manipulating genes in Drosophila 14,15. For example, recombineering, which is widely us...

Clean molecularly-defined manipulations of single genes at their endogenous loci offer an invaluable tool to study a myriad of questions relevant to eukaryotic biology. Drosophila reverse genetic techniques for generating loss-of-function alleles had proven to be challenging until Golic and colleagues introduced in vivo gene targeting using homologous recombination to Drosophila 1-3. They demonstrated that specific genomic loci could be targeted using a linear fragment of DNA from an integrated transgenic construct. This linear "donor" DNA is generated in vivo through FRT-mediated recombination (to excise the DNA from the chromosome as a circular molecule) followed by linearization with the meganuclease I-SceI. Although this methodology has been successfully used to generate a variety of defined lesions, the technique has not been easily scalable for the manipulation of numerous genes in parallel because each individual knockout construct requires distinct and custom design. For example, difficulties in seamlessly manipulating large fragments of DNA (&gt;5 kb) in vitro using classical restriction enzyme/ligation cloning or PCR, as well as the size limitations of traditional in vivo transformation vectors often interfere with the rapid creation of homologous recombination targeting vectors. To overcome these limitations, we combined the recombineering/transgenesis P[acman] system, which allows the sub-cloning and transgenesis of up to 100 kb of DNA, with the ends-out gene targeting methodology to establish an efficient and relatively rapid platform that facilitates Drosophila gene targeting.
Recombination-mediated genetic engineering (recombineering) is a powerful homologous recombination-based cloning technology 4,5. In contrast to conventional restriction enzyme/ligase cloning, recombineering is not limited by the sequence or size of the manipulated DNA. Recombineering uses a special E. coli strain that harbors recombination machinery provided by a defective &lambda; prophage 4. This technique has recently been adopted for use in Drosophila 6,7. Recombineering in Drosophila relies on a modified conditionally amplifiable bacterial artificial chromosome (BAC) vector called P[acman] 6,7. This vector carries two origins of replication: OriV, which produces high-copy number upon chemical induction for the purification of large quantities of DNA required for sequencing and embryo injection and OriS, which maintains low-copy number under basal conditions. Additionally, the P[acman] vector is equipped with a bacterial attachment (attB) site. The attB site serves as a substrate for &Phi;C31 integrase-mediated transgenesis that allows incorporation of large DNA fragments into a predetermined landing site within the Drosophila genome 8,9.
We have generated a P[acman] vector (referred to as P[acman]-KO 1.0) that can be used as a targeting vector for ends-out homologous recombination 10,11. To incorporate ends-out gene targeting technology into the system, we added two FRT and two I-SceI sites. We have also included a Gateway cassette within this modified vector to streamline the process of incorporating the homology arms into P[acman]-KO 1.0. This provides a rapid and simple way to introduce virtually any genomic region of interest into the targeting vector. In this protocol we will describe how to engineer a targeting vector using P[acman]-KO 1.0, and how to mobilize this vector in vivo to target the endogenous locus. For the purpose of this protocol we will use the RFP/Kan cassette to replace a gene of interest, but a variety of cassettes that contain an antibiotic selection marker can be used with this protocol. We have designed and successfully used a set of cassettes for gene replacement and tagging 10,11.

<table><tbody><tr><td>SW102 Recombination competent bacteria</td><td>NCI-Frederick</td><td>Recombination Bacteria (SW102, SW105 and SW106)</td><td>&lt;a href=&quot;http://ncifrederick.cancer.gov/research/brb/logon.aspx&quot; target=&quot;_blank&quot;&gt;http://ncifrederick.cancer.gov/research/brb/logon.aspx&lt;/a&gt;</td></tr><tr><td>TransforMax EPI300 electrocopmpetent &lt;em&gt;E. coli&lt;/em&gt;</td><td>Epicentre</td><td>EC300110</td><td>Includes CopyControl induction solution</td></tr><tr><td>PfuUltra II Fusion HS DNA Polymerase</td><td>Aligent Technology Inc.</td><td>600670</td><td></td></tr><tr><td>BamHI-HF</td><td>New England Biolabs</td><td>R3136S</td><td></td></tr><tr><td>Zymoclean Gel DNA Recovery Kit</td><td>Zymo Research</td><td>D4001</td><td></td></tr><tr><td>Use Gateway BP ClonaseII Enzyme kit</td><td>Invitrogen</td><td>11789-020</td><td></td></tr><tr><td>P[acman]KO1.010</td><td>Buszczak and Hiesinger Labs</td><td>Upon request</td><td></td></tr><tr><td>pENTR RFP-Kan11</td><td>Buszczak and Hiesinger Labs</td><td>Upon request</td><td></td></tr><tr><td>Flystocks</td><td>Bloomington stock center</td><td>Stock numbers: 25680, 25679</td><td>y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}11 P{70I-SceI}2B snaSco/CyO, P{hs-hid}4&lt;br/&gt; y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}23 P{70I-SceI}4A/TM3, P{hs-hid}14, Sb1</td></tr><tr><td>Electroporation machine</td><td>Biorad GenePulser Xcell with PC module</td><td>165-2662</td><td></td></tr><tr><td>Cuvettes</td><td>Fisher Brand</td><td>#FB101</td><td></td></tr></tbody></table>

recombineering homologous recombination constructs in drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Amplification of the LA and RA homology arms should produce 500 bp products and the PCR-SOE reaction should yield a 1.0 kb product (Sections 2.1-2.4; Figure 2). The BP reaction performed in section 2.5 is typically very efficient and bacterial transformation of the product yields 5-100 colonies on average. Nearly all the colonies tested with PCR check show the expected PCR product.
During the first round of recombineering (Section 3) expect to get 20-40 colonies after transfor...

Watch this Scientific Journal Video about Recombineering Homologous Recombination Constructs in Drosophila at JoVE.com

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

recombineering-homologous-recombination-constructs-in-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.1K Views.  University of Texas Southwestern Medical Center at Dallas. Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Video: Recombineering Homologous Recombination Constructs in Drosophila

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Cancer Research

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Genetics

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Biochemistry

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

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

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 patterns.

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Developmental Biology

Cryopreservation of PGCs for Long-Term Preservation of Drosophila Strains

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15&#160;agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 &#177; 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 &#177; 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

Author Spotlight: PGC Cryopreservation &#8211; An Alternative Approach Towards Long-Term Preservation of Drosophila Strains 

A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and ...

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

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.

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.

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

Neuroscience

Drosophila Development and Reproduction

One of the many reasons that make Drosophila an extremely valuable organism is that the molecular, cellular, and genetic foundations of development are highly conserved between flies and higher eukaryotes such as humans. Drosophila progress through several developmental stages in a process known as the life cycle and each stage provides a unique platform for developmental research. This video introduces each stage of the Drosophila life cycle and details the physical characteristics and major developmental events that occur during each stage. Next, the video discusses the genetic regulation of pattern formation, which is important for establishing the body plan of the organism and specifying individual tissues and organs. In addition, this video gives an overview of Drosophila reproduction, and how to use the reproductive characteristics of Drosophila to set up a genetic cross. Finally, we discuss examples of how the principles of Drosophila development and reproduction can be applied to research. These applications include RNA interference, behavioral assays of mating behaviors, and live imaging techniques that allow us to visualize development as a dynamic process. Overall, this video highlights the importance of understanding development and reproduction in Drosophila, and how this knowledge can be used to understand development in other organisms.

Development and Reproduction of Drosophila melanogaster

One of the many reasons that make Drosophila an extremely valuable organism is that the molecular, cellular, and genetic foundations of development are ...

Recombineering Homologous Recombination Constructs in Drosophila

Summary

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

Drosophila Development and Reproduction