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 ...

The main goal of this protocol is to use the power of forward genetics to identify genes which when dysregulated lead to neurodegeneration. The main advantage of this technique is that it provides an unbiased and straightforward approach for the identification of neuroprotective genes which might be more difficult to perform in mammalian models. This technique can be used to identify novel genes implicated in the process of neurodegeneration which is associated with many disease conditions such as Alzheimer's and Parkinson's disease.In addition to identifying neuroprotective genes, the forward genetic approach can be used to discover genes involved in other biological processes such as neuronal function and transmission. To begin, collect homozygous flies from lines belonging to the ENU mutagenized collection of Drosophila mutants mapped to the second chromosome. Flip the flies aged 10 to 12 days into a testing chamber without anesthesia one line at a time and allow them to recover for five minutes.With the five centimeter line mark side down, forcibly tap the chamber three times on a mouse pad placed on a solid surface so that all flies begin the assay at the bottom. Observe fly locomotion over a period of 10 seconds. Record the number of flies that reach or pass the five centimeter line within this time as well as the total number of flies tested.Wait one minute before starting a second trial. Repeat a minimum of three trial replicates per line. After anesthetizing the flies on a carbon dioxide pad, sever fly heads with a surgical blade and use a paintbrush to gently place them in one milliliter of Carnoy's fixative in a 1.5 milliliter microcentrifuge tube.Then store the tube at four degrees Celsius overnight. The following day, make sure the heads are sunk in the fixative solution. Under a fume hood, use a P1000 pipette to replace the fixative solution with one milliliter of 70%ethanol and maintain the samples at four degrees Celsius for future analysis.Using a Pasteur pipette, transfer the fixed heads into microbiopsy cassettes and close the cassettes. Send the cassettes to a histology facility for processing or place them in an automated tissue processing machine if available on site. Run the program to dehydrate, clear and infiltrate the heads with paraffin.Then transfer the cassettes into a paraffin embedding station with paraffin heated at 60 degrees Celsius and place metal base molds that fit the cassettes on the heated station. Use fine forceps to orient the heads such that they face the top in the base mold. Proper head orientation is critical for the histology analysis.After hardening, trim each paraffin block to minimize the surface that will be sectioned. Cut five micrometer sections of each block on the microtome. Next, place the sectioned paraffin ribbons in a heated water bath at 35 degrees Celsius for a maximum of five minutes.Immerse a polylysine-coated slide in the heated water bath and use a wood applicator to place the sectioned ribbons on the slide. Let the slides air dry overnight at room temperature. The next day, use a slide warmer to heat the slides for 15 minutes at 63 degrees Celsius.Under a fume hood, place the slides on a slide staining rack and place the rack in HistoChoice for five minutes twice followed by several ethanol and distilled water bath treatments according to the manuscript. Transfer the rack into a container filled with Harris Hematoxylin for two to five minutes to stain. Then take the rack out of the Hematoxylin solution and place it under running tap water for 10 minutes before the following staining steps.After staining, using forceps, take the slides out of the HistoChoice or Xylene solution. Drip a thin layer of permanent mounting medium on the sample and gently place a coverslip on the top. Avoid formation of bubbles.After the mounting medium is hardened, place the slide under a light microscope to obtain images of representative brain sections at approximately midbrain. First, prepare a food containing vial for generating heterozygous female flies. After carbon dioxide anesthesia, use a paintbrush to transfer five males of the mutant line and 10 virgin females of the sternopleural jammed lobe and pin over Curly O line into the vial to make a cross.Place the vial at 25 degrees Celsius. After 10 to 12 days, collect at least 15 virgin female progeny carrying the mutated chromosome and the marker chromosome. Cross them to five males from a balanced line that carries another visible dominant marker Curly O over scutoid or equivalent.After 10 to 12 days, collect the heterozygous male progeny carrying either scutoid or Curly O and the potentially recombined chromosome from the cross. Individually mate them to virgin females from the stock carrying the mutated chromosome. Ten to 12 days later, collect the progeny from the last cross into new food containing vials and age the flies at 29 degrees Celsius to 10 to 14 days.Perform histology on fly heads and repeat for progeny of each individual cross. With this information, perform deficiency mapping on narrowed region of the chromosome to refine the location of the recessive mutation. Each deficiency line has a deletion of different region of the chromosomes allowing them to be used for complementation testing.Next, cross lines from the Drosophila deficiency kit for the second chromosome and look for non-complementation of the phenotype of the interest. To obtain reference for DNA sequence analysis, download a FASTA format file containing the genomic consensus sequence of the BRAT gene from FlyBase or use a file containing results for BRAT sequence of a genetic background control, for example the originally mutagenized Drosophila line. Open sequencing files in the A Plasmid Editor.Go to Edit and click Align Sequences. Using the computer's mouse, select all sequences to compare. In the drop menu, indicate the reference sequence for this alignment.Inspect the sequenced region for nucleotide changes in comparison with the reference sequence. If desired, save the alignment on a computer in RTF format by clicking on Text then Save. Perform the rescue experiment by collecting F1 progeny from each cross.Carefully select away marked balancers. Age the flies on Drosophila food medium at 29 degrees Celsius and perform histology analysis on brains to look for neurodegeneration as previously. To perform age-dependent analysis of neurodegeneration in 867 mutants, age the flies to five, 15 and 25 days and perform subsequent histology analysis.This protocol presents a forward genetic approach to screen through a collection of chemically mutagenized flies using a climbing assay. Among 235 homozygous lines, about 37%of the tested lines exhibited a climbing pass rate below 50%when tested at the age of 10 to 12 days. Subsequent histological screen on 51 of the lines exhibiting the lowest climbing pass rate revealed that 29 of these lines showed visible appearance of holes in the brain neuropil ranging from mild to severe indicative of neurodegeneration.The neurodegeneration phenotype in 867 flies is recessive because the brains of heterozygous 867 flies that have been out-crossed to a wild type strain were comparable to those of controls. The deficiency line Df24116 does not complement the 867 mutant phenotype based on the climbing assay. Histological verification shows the deficiency line Df24116 does not complement the 867 neurodegeneration phenotype whereas Df9296 does.Examining the brain phenotype of 867 mutants crossed to additional deficiency lines confirmed that the mutation is contained in the region of the genome that includes the gene BRAT. It's important to remember during this procedure to work as rigorously and precisely as possible as this protocol combines several steps which if executed improperly can affect the identification of mutants with neurodegeneration in candidate genes. Depending on the genes identified, subsequent analysis can include testing additional ovules of the gene of the interest or interactions with genes that play a role in known pathways of neurodegeneration.During the histology procedure, remember to handle the Carnoy's fixative solution with caution since it contains chloroform. Wear gloves and use with adequate ventilation ideally under the fume hood. Similarly, perform all histology staining under the fume hood.

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

Research

JoVE Journal

Genetics

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 replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

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

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.

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 of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

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

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

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

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

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.

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

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

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

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

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

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

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

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

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)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

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

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the 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 incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

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

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

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

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 ...

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 agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

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

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study 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 ...