Name: in vivo forward genetic screen to identify novel neuroprotective genes in drosophila melanogaster
Uploaded: 2019-07-11T14:30:00
Description: Watch this Scientific Journal Video about In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster at JoVE.com

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.

1. Preparation and Aging of Flies
<ol>
	<li>Obtain or generate6 a collection of Drosophila mutants that will be used for the genetic screen. Here, ENU-mutagenized lines mapped to the second chromosome and balanced over CyO are used.</li>
	<li>Amplify experimental genotype lines in an incubator set at 25 &#176;C, 12 h light/dark cycle on cornmeal-molasses medium.</li>
	<li>Collect around 20 homozygous progeny between 0-2 days of adult eclosion from each experimental genotype. El...

<ol>
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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.

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			<td colspan="4" height="21" style="height:21px;width:743px;">Major equipment</td>
		</tr>
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			<td height="21" style="height:21px;width:255px;">Fume hood for histology</td>
			<td style="width:195px;"></td>
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			<td></td>
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			<td height="42" style="height:42px;width:255px;">Light Microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:123px;">Eclipe E100</td>
			<td style="width:171px;">Preferred objective for imaging is X20</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Imaging software</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:123px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microscope Camera</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:123px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Thermal cycler</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:123px;"></td>
			<td></td>
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			<td colspan="4" height="21" style="height:21px;width:743px;">Fly pushing and climbing assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">VWR&#174; Drosophila Vial, Narrow</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">75813-160</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">VWR&#174; General-Purpose Laboratory Labeling Tape</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">89097-912</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:255px;">Standard mouse pad</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Stereoscope</td>
			<td style="width:195px;">Motic</td>
			<td style="width:123px;"></td>
			<td>Model SMZ-168</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:255px;">CO2 anesthesia station (Blowgun, foot valve, Ultimate Flypad)</td>
			<td style="width:195px;">Genesee Scientific</td>
			<td style="width:123px;">54-104, 59-121, 59-172</td>
			<td>Doesn&#8217;t iinclude CO2 tank</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fine-Tip Brushes</td>
			<td style="width:195px;">SOLO HORTON BRUSHES, INC.</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Drosophila Incubator</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">89510-750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:743px;">Gene mapping</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">CantonS</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td>9517</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">w1118</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td>5905</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">yw&#160;</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td align="right">6599</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">Drosophila line used for recombination mapping</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">3227</td>
			<td style="width:171px;">Genotype: wg[Sp-1] J[1] L[2] Pin[1]/CyO, P{ry[+t7.2]=ftz/lacB}E3</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">CyO/sno[Sco]&#160;</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">2555</td>
			<td style="width:171px;">Drosophila balancer line used for recombination mapping</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Deficiency Kit for chromosome 2L</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">DK2L</td>
			<td>Cook et al., 2012</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Histology analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:255px;">Ethanol, (100%)</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">A4094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloroform</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glacial Acetic Acid</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">A38-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fisherbrand&#8482;&#160;Premium Microcentrifuge Tubes: 1.5mL</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Histochoice clearing agent 1X</td>
			<td style="width:195px;">VWR Life Sciences</td>
			<td style="width:123px;">97060-934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Harris Hematoxylin</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">95057-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Eosin</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">95057-848</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Thermo Scientific&#8482;&#160;Richard-Allan Scientific&#8482; Mounting Medium</td>
			<td style="width:195px;">Thermo Scientific&#8482; 4112</td>
			<td style="width:123px;">22-110-610</td>
			<td>CyO/sna[Sco]</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">Unifrost Poly-L-Lysine microscope slides, 75x25x1mm, EverMark Select Plus</td>
			<td style="width:195px;">Azer Scientific</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fisherbrand&#8482;&#160;Cover Glasses: Rectangles</td>
			<td style="width:195px;">Fisherbrand</td>
			<td style="width:123px;">12-545M</td>
			<td>Dimensions: 24x60 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Traceable timer</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Slide Warmer</td>
			<td style="width:195px;">Barnstead International</td>
			<td style="width:123px;"></td>
			<td>model no. 26025</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Slide tray and racks</td>
			<td style="width:195px;">DWK Life Sciences</td>
			<td style="width:123px;"></td>
			<td>Rack to hold 20 slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fisherbrand&#8482;&#160;General-Purpose Extra-Long Forceps</td>
			<td style="width:195px;">Fisherbrand</td>
			<td style="width:123px;">10-316A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Kimwipes&#8482;</td>
			<td style="width:195px;">Kimberly-Clark&#8482; Professional&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">6 inch Puritan applicators</td>
			<td style="width:195px;">Hardwood Products Company, Guilford, Maine</td>
			<td style="width:123px;">807-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">VWR&#174; Razor Blades</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tupperware or glass containers for histology liquids</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td>16 + 1 for running water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">High Profile Coated Microtome Blades</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">95057-834</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Corning&#8482;&#160;Round Ice Bucket with Lid, 4L</td>
			<td style="width:195px;">Corning&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Beaker</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td>Or other container for ice water and cassettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tissue Bath</td>
			<td style="width:195px;">Precision Scientific Company</td>
			<td style="width:123px;">66630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtome</td>
			<td style="width:195px;">Leica Biosystems</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Molecular analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Wizard&#174; SV Gel and PCR Clean-Up System</td>
			<td style="width:195px;">Promega</td>
			<td style="width:123px;">A9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ex Taq DNA polymerase</td>
			<td style="width:195px;">TaKaRa</td>
			<td style="width:123px;"></td>
			<td>5 U/&#956;l</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Invitrogen&#8482;&#160;SYBR&#8482; Safe&#8482; DNA Gel Stain&#160;</td>
			<td style="width:195px;">&#160;Invitrogen&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">UltraPure&#8482; Agarose</td>
			<td style="width:195px;">&#160;Invitrogen&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1 Kb Plus DNA Ladder</td>
			<td style="width:195px;">&#160;Invitrogen&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">ApE-A plasmid Editor software</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td style="width:171px;">Available for free download</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Statistical analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">R software package</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Further analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">y[1] w[*]; wg[Sp-1]/CyO; Dr[1]/TM3, Sb[1]</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">59967</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 (...

Watch this Scientific Journal Video about In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster at JoVE.com

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

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

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 ...

Research

JoVE Journal

Genetics

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

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

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Quantifying Abdominal Pigmentation in Drosophila melanogaster

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

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

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

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

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

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

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

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

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

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 ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...