Name: In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2019-07-11T14:30:00.000+00:00
Duration: 10 min
Description: Discover a forward genetic screen to identify neuroprotective genes in Drosophila melanogaster using climbing assays and histology. This method reveals novel targets for neurodegenerative disease research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

We are particularly grateful to Dr. Barry Ganetzky, in who&#39;s lab the genetic screen was performed, allowing the identification and characterization of brat as a neuroprotective gene. We thank Dr. Steven Robinow for kindly providing the collection of ENU-mutagenized flies used in the genetic screen presented in this article. We thank the members of Ganetzky lab, Drs. Grace Boekhoff-Falk and David Wassarman for helpful discussions throughout the duration of this project, Ling Ling Ho and Bob Kreber for technical assistance, Dr. Aki Ikeda for the use of his microtome facility at the University of Wisconsin and Dr. Kim Lackey and the Optical Analysis Facility at the University of Alabama.

<ol><li>Frade, J. M., Ovejero-Benito, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+cell+cycle%3A+the+neuron+itself+and+its+circumstances%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Neuronal cell cycle: the neuron itself and its circumstances.</a> Cell Cycle. 14 (5), 712-720 (2015).</li>
<li>Aranda-Anzaldo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+post-mitotic+state+in+neurons+correlates+with+a+stable+nuclear+higher-order+structure%5BTitle%5D)+AND+%22Communicative+%26+Integrative+Biology%22%5BJournal%5D)">The post-mitotic state in neurons correlates with a stable nuclear higher-order structure.</a> Communicative & Integrative Biology. 5 (2), 134-139 (2012).</li>
<li>Haelterman, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+identification+of+chemically+induced+mutations+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Large-scale identification of chemically induced mutations in Drosophila melanogaster.</a> Genome Res. 24 (10), 1707-1718 (2014).</li>
<li>Moresco, E. M., Li, X., Beutler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Going+forward+with+genetics%3A+recent+technological+advances+and+forward+genetics+in+mice%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">Going forward with genetics: recent technological advances and forward genetics in mice.</a> The American Journal of Pathology. 182 (5), 1462-1473 (2013).</li>
<li>St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+art+and+design+of+genetic+screens%3A+Drosophila+melanogaster%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">The art and design of genetic screens: Drosophila melanogaster.</a> Nature Reviews Genetics. 3 (3), 176-188 (2002).</li>
<li>Greenspan, R. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreenspan%2C+RJ+%22Fly+pushing%3A+The+theory+and+practice+of+Drosophila+genetics%22">Fly pushing: The theory and practice of Drosophila genetics</a>. , Cold Spring Harbor Laboratory Press. Second edition (2004).</li>
<li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Research. 11 (6), 1114-1125 (2001).</li>
<li>Rubin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+genomics+of+the+eukaryotes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Comparative genomics of the eukaryotes.</a> Science. 287 (5461), 2204-2215 (2000).</li>
<li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+to+assay+Drosophila+behavior%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Methods to assay Drosophila behavior.</a> Journal of Visualized Experiments. (61), (2012).</li>
<li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assaying+locomotor%2C+learning%2C+and+memory+deficits+in+Drosophila+models+of+neurodegeneration%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> Journal of Visualized Experiments. (49), (2011).</li>
<li>Karres, J. S., Hilgers, V., Carrera, I., Treisman, J., Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+conserved+microRNA+miR-8+tunes+atrophin+levels+to+prevent+neurodegeneration+in+Drosophila%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The conserved microRNA miR-8 tunes atrophin levels to prevent neurodegeneration in Drosophila.</a> Cell. 131 (1), 136-145 (2007).</li>
<li>Helfrich, C., Engelmann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian-Rhythm+of+the+Locomotor-Activity+in+Drosophila-Melanogaster+and+Its+Mutants+Sine+Oculis+and+Small+Optic+Lobes%5BTitle%5D)+AND+%22Physiological+Entomology%22%5BJournal%5D)">Circadian-Rhythm of the Locomotor-Activity in Drosophila-Melanogaster and Its Mutants Sine Oculis and Small Optic Lobes.</a> Physiological Entomology. 8 (3), 257-272 (1983).</li>
<li>R Development Core Team. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22R%3A+A+language+and+environment+for+statistical+computing%22">R: A language and environment for statistical computing</a>. , Foundation for Statistical Computing. (2008).</li>
<li>Bokel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EMS+screens+%3A+from+mutagenesis+to+screening+and+mapping%5BTitle%5D)+AND+%22Methods+in+Molecular+Bioogyl%22%5BJournal%5D)">EMS screens : from mutagenesis to screening and mapping.</a> Methods in Molecular Bioogyl. 420, 119-138 (2008).</li>
<li>Lindsley, D. L., Zimm, G. G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLindsley%2C+DL+%22The+Genome+of+Drosophila+Melanogaster%22">The Genome of Drosophila Melanogaster</a>. , (1992).</li>
<li>Cook, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+generation+of+chromosomal+deletions+to+provide+extensive+coverage+and+subdivision+of+the+Drosophila+melanogaster+genome%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome.</a> Genome Biology. 13 (3), R21(2012).</li>
<li>Loewen, C., Boekhoff-Falk, G., Ganetzky, B., Chtarbanova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Mutation+in+Brain+Tumor+Causes+Both+Neural+Over-Proliferation+and+Neurodegeneration+in+Adult+Drosophila%5BTitle%5D)+AND+%22Genes+Genomes+Genetics+G3+%28Bethesda%29%22%5BJournal%5D)">A Novel Mutation in Brain Tumor Causes Both Neural Over-Proliferation and Neurodegeneration in Adult Drosophila.</a> Genes Genomes Genetics G3 (Bethesda). 8 (10), 3331-3346 (2018).</li>
<li>Gloor, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+I+repressors+of+P+element+mobility%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Type I repressors of P element mobility.</a> Genetics. 135 (1), 81-95 (1993).</li>
<li>Komori, H., Xiao, Q., McCartney, B. M., Lee, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+tumor+specifies+intermediate+progenitor+cell+identity+by+attenuating+beta-catenin%2FArmadillo+activity%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Brain tumor specifies intermediate progenitor cell identity by attenuating beta-catenin/Armadillo activity.</a> Development. 141 (1), 51-62 (2014).</li>
<li>Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+swiss+cheese+mutant+causes+glial+hyperwrapping+and+brain+degeneration+in+Drosophila%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila.</a> The Journal of Neuroscience. 17 (19), 7425-7432 (1997).</li>
<li>Kang, K. H., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+neural+stem+cell+self-renewal+and+differentiation+in+Drosophila%5BTitle%5D)+AND+%22Cell+and+Tissue+Research%22%5BJournal%5D)">Control of neural stem cell self-renewal and differentiation in Drosophila.</a> Cell and Tissue Research. 359 (1), 33-45 (2015).</li>
<li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+Gene-Expression+as+a+Means+of+Altering+Cell+Fates+and+Generating+Dominant+Phenotypes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Targeted Gene-Expression as a Means of Altering Cell Fates and Generating Dominant Phenotypes.</a> Development. 118 (2), 401-415 (1993).</li>
<li>Loewen, C. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mito-Nuclear+Interactions+Affecting+Lifespan+and+Neurodegeneration+in+a+Drosophila+Model+of+Leigh+Syndrome%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome.</a> Genetics. 208 (4), 1535-1552 (2018).</li>
<li>Cao, Y., Chtarbanova, S., Petersen, A. J., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dnr1+mutations+cause+neurodegeneration+in+Drosophila+by+activating+the+innate+immune+response+in+the+brain%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Dnr1 mutations cause neurodegeneration in Drosophila by activating the innate immune response in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (19), E1752-E1760 (2013).</li>
<li>Lessing, D., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintaining+the+brain%3A+insight+into+human+neurodegeneration+from+Drosophila+melanogaster+mutants%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Maintaining the brain: insight into human neurodegeneration from Drosophila melanogaster mutants.</a> Nature Reviews Genetics. 10 (6), 359-370 (2009).</li>
<li>Peng, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+Polo+ameliorates+APP-induced+Alzheimer%27s+disease-like+symptoms+in+Drosophila%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Loss of Polo ameliorates APP-induced Alzheimer's disease-like symptoms in Drosophila.</a> Scientific Reports. 5, 16816(2015).</li>
<li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+mutagens%2C+transposons%2C+and+transgenes+to+interrogate+gene+function+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster.</a> Methods. 68 (1), 15-28 (2014).</li>
<li>Gonzalez, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+Genome+Sequencing+and+a+New+Bioinformatics+Platform+Allow+for+Rapid+Gene+Identification+in+D.+melanogaster+EMS+Screens%5BTitle%5D)+AND+%22Biology+%28Basel%29%22%5BJournal%5D)">Whole Genome Sequencing and a New Bioinformatics Platform Allow for Rapid Gene Identification in D. melanogaster EMS Screens.</a> Biology (Basel). 1 (3), 766-777 (2012).</li>
<li>Palladino, M. J., Hadley, T. J., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature-sensitive+paralytic+mutants+are+enriched+for+those+causing+neurodegeneration+in+drosophila%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Temperature-sensitive paralytic mutants are enriched for those causing neurodegeneration in drosophila.</a> Genetics. 161 (3), 1197-1208 (2002).</li>
</ol>

The authors declare no conflicts of interest.

Forward genetic screens in Drosophila have been an effective approach to identify genes involved in different biological processes, including age-dependent neuroprotection5,23,24,25. Using this strategy, we were successful in identifying brat as a novel neuroprotective gene17.
One critical step in this protocol involves the p...

Neurons are for the most part post-mitotic and incapable of dividing1,2. In most animals, neuroprotective mechanisms exist to maintain these cells throughout the organism&#39;s lifespan, especially at old age when neurons are most vulnerable to damage. Genes underlying these mechanisms can be identified in mutants exhibiting neurodegeneration, a phenotypic indicator for the loss of neuroprotection, using a forward genetic protocol. Forward genetic screens using chemical mutagens such as ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU) are particularly useful due to the random point mutations they induce, resulting in an inherently unbiased approach that has shed light on numerous gene functions in eukaryotic model organisms3,4,5 (in contrast, X-ray mutagenesis creates DNA breaks and can result in rearrangement rather than point mutations6).
The common fruit fly Drosophila melanogaster is an ideal subject for these screens due to its high quality, well annotated genome sequence, its long history as a model organism with highly developed genetic tools, and most significantly, its shared evolutionary history with humans7,8. A limiting factor in the applicability of this protocol is early lethality caused by the mutated genes, which would prevent testing at old age9. However, for non-lethal mutations, a climbing assay, which takes advantage of negative geotaxis, is a simple, although extensive, method of quantifying impaired motor functioning10. To exhibit sufficient locomotor reactivity, flies depend on neural functions to determine direction, sense its position, and coordinate movement. The inability of flies to sufficiently climb in response to stimuli can therefore indicate neurological defects11. Once a particular defective climbing phenotype is identified, further testing using a secondary screen such as histological analysis of brain tissue, can be used to identify neurodegeneration in climbing-defective flies. Subsequent gene mapping can then be used to reveal the genomic region on the chromosome carrying the defective neuroprotective gene of interest. To narrow down the chromosomal region of interest, meiotic mapping using mutant fly lines carrying dominant marker genes with known locations on the chromosome can be performed. The marker genes serve as a reference point for the mutation as the frequency of recombination between two loci provides a measurable distance that can be used to map the approximate location of a gene. Finally, crossing the mutant lines with lines carrying balanced deficiencies on the meiotically mapped chromosomal region of interest creates a complementation test in which the gene of interest can be verified if its known phenotype is expressed5. Polymorphic nucleotide sequences in the identified gene, possibly resulting in an altered amino acid sequences, can be evaluated by sequencing the gene and comparing it to the Drosophila genome sequence. Subsequent characterization of the gene of interest can include testing of additional mutant alleles, mutation rescue experiments and examination of additional phenotypes.

<table><tbody><tr><td>&lt;strong&gt;Major equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fume hood for histology</td><td></td><td></td><td></td></tr><tr><td>Light Microscope</td><td>Nikon</td><td>Eclipe E100</td><td>Preferred objective for imaging is 20x</td></tr><tr><td>Imaging software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Microscope Camera</td><td>Nikon</td><td></td><td></td></tr><tr><td>Thermal cycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fly pushing and climbing assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>VWR&amp;reg; Drosophila Vial, Narrow</td><td>VWR</td><td>75813-160</td><td></td></tr><tr><td>VWR&amp;reg; General-Purpose Laboratory Labeling Tape</td><td>VWR</td><td>89097-912</td><td></td></tr><tr><td>Standard mouse pad</td><td></td><td></td><td></td></tr><tr><td>Stereoscope</td><td>Motic</td><td></td><td>Model SMZ-168</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; anesthesia station (Blowgun, foot valve, Ultimate Flypad)</td><td>Genesee Scientific</td><td>54-104, 59-121, 59-172</td><td>Doesn&amp;rsquo;t iinclude CO2 tank</td></tr><tr><td>Fine-Tip Brushes</td><td>SOLO HORTON BRUSHES, INC.</td><td></td><td></td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; Incubator</td><td>VWR</td><td>89510-750</td><td></td></tr><tr><td>&lt;strong&gt;Gene mapping&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CantonS</td><td>Bloomington Drosophila Stock Center</td><td>9517</td><td></td></tr><tr><td>w&lt;sup&gt;1118&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>5905</td><td></td></tr><tr><td>yw&amp;nbsp;</td><td>Bloomington Drosophila Stock Center</td><td>6599</td><td></td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; line used for recombination mapping</td><td>Bloomington Drosophila Stock Center</td><td>3227</td><td>Genotype: wg[Sp-1] J[1] L[2] Pin[1]/CyO, P{ry[+t7.2]=ftz/lacB}E3</td></tr><tr><td>CyO/sno[Sco]&amp;nbsp;</td><td>Bloomington Drosophila Stock Center</td><td>2555</td><td>Drosophila balancer line used for recombination mapping</td></tr><tr><td>Deficiency Kit for chromosome 2L</td><td>Bloomington Drosophila Stock Center</td><td>DK2L</td><td>Cook et al., 2012</td></tr><tr><td>&lt;strong&gt;Histology analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol, (100%)</td><td>Thermo Fischer Scientific</td><td>A4094</td><td></td></tr><tr><td>Chloroform</td><td>Thermo Fischer Scientific</td><td>C298-500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Thermo Fischer Scientific</td><td>A38-500</td><td></td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;Premium Microcentrifuge Tubes: 1.5 mL</td><td>Thermo Fischer Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Histochoice clearing agent 1x</td><td>VWR Life Sciences</td><td>97060-934</td><td></td></tr><tr><td>Harris Hematoxylin</td><td>VWR</td><td>95057-858</td><td></td></tr><tr><td>Eosin</td><td>VWR</td><td>95057-848</td><td></td></tr><tr><td>Thermo Scientific&amp;trade;&amp;nbsp;Richard-Allan Scientific&amp;trade; Mounting Medium</td><td>Thermo Scientific&amp;trade; 4112</td><td>22-110-610</td><td>CyO/sna[Sco]</td></tr><tr><td>Unifrost Poly-L-Lysine microscope slides, 75 mm x 25 mm x 1 mm, EverMark Select Plus</td><td>Azer Scientific</td><td></td><td></td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;Cover Glasses: Rectangles</td><td>Fisherbrand</td><td>12-545M</td><td>Dimensions: 24 mm x 60 mm</td></tr><tr><td>Traceable timer</td><td>VWR</td><td></td><td></td></tr><tr><td>Slide Warmer</td><td>Barnstead International</td><td></td><td>model no. 26025</td></tr><tr><td>Slide tray and racks</td><td>DWK Life Sciences</td><td></td><td>Rack to hold 20 slides</td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;General-Purpose Extra-Long Forceps</td><td>Fisherbrand</td><td>10-316A</td><td></td></tr><tr><td>Kimwipes&amp;trade;</td><td>Kimberly-Clark&amp;trade; Professional&amp;nbsp;</td><td></td><td></td></tr><tr><td>6 inch Puritan applicators</td><td>Hardwood Products Company, Guilford, Maine</td><td>807-12</td><td></td></tr><tr><td>VWR&amp;reg; Razor Blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Tupperware or glass containers for histology liquids</td><td></td><td></td><td>16 + 1 for running water</td></tr><tr><td>High Profile Coated Microtome Blades</td><td>VWR</td><td>95057-834</td><td></td></tr><tr><td>Corning&amp;trade;&amp;nbsp;Round Ice Bucket with Lid, 4 L</td><td>Corning&amp;trade;</td><td></td><td></td></tr><tr><td>Beaker</td><td></td><td></td><td>Or other container for ice water and cassettes</td></tr><tr><td>Tissue Bath</td><td>Precision Scientific Company</td><td>66630</td><td></td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Molecular analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Wizard&amp;reg; SV Gel and PCR Clean-Up System</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Ex Taq DNA polymerase</td><td>TaKaRa</td><td></td><td>5 U/&amp;mu;l</td></tr><tr><td>Invitrogen&amp;trade;&amp;nbsp;SYBR&amp;trade; Safe&amp;trade; DNA Gel Stain&amp;nbsp;</td><td>&amp;nbsp;Invitrogen&amp;trade;</td><td></td><td></td></tr><tr><td>UltraPure&amp;trade; Agarose</td><td>&amp;nbsp;Invitrogen&amp;trade;</td><td></td><td></td></tr><tr><td>1 Kb Plus DNA Ladder</td><td>&amp;nbsp;Invitrogen&amp;trade;</td><td></td><td></td></tr><tr><td>ApE-A plasmid Editor software</td><td></td><td></td><td>Available for free download</td></tr><tr><td>&lt;strong&gt;Statistical analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>R software package</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Further analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>y[1] w[*]; wg[Sp-1]/CyO; Dr[1]/TM3, Sb[1]</td><td>Bloomington Drosophila Stock Center</td><td>59967</td><td></td></tr></tbody></table>

in vivo forward genetic screen to identify novel neuroprotective genes in drosophila melanogaster

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

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

In this aerticle, we present the steps used to identify the gene brain tumor (brat) as playing a role in maintenance of neuronal integrity (e.g., neuroprotection) in adult flies17; a methodology that can be used to identify genes involved in neuroprotection. We used a forward genetic approach (the strategy is outlined in Figure 1A) to screen through a collection of chemically mutagenized flies using a climbing assay (...

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

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

in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective

Research

JoVE Journal

Genetics

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

This video describes a simple method to quantify fly locomotion and shows the climbing assay performed.  The fly climbing assay is a general test of motor function which relies on a negative geotaxis response.

Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

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.

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

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 larvae.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI)  to adult Drosophila and examining the resulting neurogenesis.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The protocol presents a high-throughput, cost-effective method for assessing neurotoxicity in the fruit fly through the quantification of locomotor dysfunction as an alternative to traditional mammalian models. It aims to evaluate the neurotoxic effects of pharmaceutical compounds, environmental agents, or genetic modifications using a sensitive, reproducible, and ethically favorable model.

Behavioral Analysis of Locomotor Dysfunction in Drosophila melanogaster as a Readout for Neurotoxicity

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Discover a forward genetic screen to identify neuroprotective genes in Drosophila melanogaster using climbing assays and histology. This method reveals novel targets for neurodegenerative disease research.

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

Explore More Videos