Name: genetic screen for identification of multicopy suppressors in schizosaccharomyces pombe
Uploaded: 2022-09-13T13:00:00.000+00:00
Duration: 13 min 22 s
Description: Watch this Scientific Journal Video about Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe at JoVE.com

This work was funded by a research grant from the Department of Biotechnology, Government of India (Grant No. BT/PR12568/BRB/10/1369/2015) to Nimisha Sharma. The authors thank Prof. Charles Hoffman (Boston College, USA) for the gift of the S. pombe cDNA library and Prof. Susan Forsburg for the yeast plasmids.

1.&#160;Transformation of the cDNA library into the query S. pombe mutant strain to screen for multicopy suppressors
NOTE: The Standard Lithium-Acetate method27 was followed to transform the S. pombe cDNA library into the query S. pombe ell1&#916;&#160;strain with a few modifications:
<ol>
	<li>Grow the S. pombe ell1&#916;&#160;strain at 32 &#176;C on a YE medium (Table 1) plate supplemented with 225 &#181;g/mL each of adenine, leucine, and uracil. Inoculate a loop full of inoculum of ell1&#916;&#160;strain from the above plate in 15-20 mL of YE medium supplemented with 225 &#181;g/mL each of adenine, leucine, and uracil. Incubate it overnight at 32 &#176;C with shaking (200-250 rpm).</li>
	<li>Next day, dilute the overnight culture in 100 mL of fresh YE medium with the desired supplements to an OD600 nm (optical density at 600 nm) of 0.3 and incubate it at 32 &#176;C with shaking at 200-250 rpm until the OD600 nm reaches mid-log phase.</li>
	<li>Divide the 100 mL culture into two 50 mL centrifuge tubes, followed by centrifugation at room temperature for 10 min at 4,000 &#215; g.</li>
	<li>Discard the supernatant and wash the cell pellet with 1 mL of sterile water, i.e., resuspend the cells in 1 mL of sterile water and centrifuge at 4,000 &#215; g for 5 min at room temperature.</li>
	<li>Discard the supernatant and wash each cell pellet with 1 mL of 1x LiAc-TE (100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) solution as described in step 1.4.</li>
	<li>Remove the supernatant and resuspend each cell pellet in 250 &#181;L of 1x LiAc-TE. Use these S. pombe competent cells (500 &#181;L) for transformation with the DNA of interest. Transfer 125 &#181;L of the competent cells into four different sterile microcentrifuge tubes for subsequent transformation steps.</li>
	<li>Boil carrier DNA from Salmon testes or herring sperm for 1 min and immediately place on ice. For each transformation, add 10 &#181;L of 10 mg/mL denatured, single-stranded carrier DNA to the microcentrifuge tube containing 125 &#181;L of the competent cells, followed by gentle mixing using a micropipette tip. Subsequently, add 50 &#181;g of the S. pombe cDNA library to the competent cells-carrier DNA mixture-containing microcentrifuge tube.</li>
	<li>Incubate the microcentrifuge tubes at room temperature for 10 min, and then add 260 &#181;L of polyethylene glycol-lithium acetate solution (40% [w/v] PEG 4,000, 100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to the tubes and mix gently with the help of a micropipette tip.</li>
	<li>Incubate the microcentrifuge tubes containing the transformation mixture at 32 &#176;C for 2 h without shaking. Add 43 &#181;L of prewarmed dimethyl sulfoxide (DMSO) to the microcentrifuge tube and mix gently. Subsequently, expose the tubes to heat shock at 42 &#176;C for 5 min.</li>
	<li>Pellet the cells at 4,000 &#215; g for 5 min at room temperature. Discard the supernatant and remove the residual PEG- LiAc-TE solution with the help of a micropipette tip.</li>
	<li>Resuspend the cells in 100 &#181;L of sterile water and plate on one EMM2 (Table 1) plate (150 mm) with required supplements and 0.2 &#181;g/mL of 4-NQO.</li>
	<li>Incubate the plates at 32 &#176;C for 5-6 days to allow colonies to appear on the plates. Streak the obtained colonies on the same medium plate in the presence of 0.2 &#181;g/mL of 4-NQO and use for further screening.</li>
	<li>For one library transformation experiment, use 500 &#181;L of competent cells (125 &#181;L of competent cells x 4 microcentrifuge tubes) for transformation as described in steps 1.8 to 1.14 above.</li>
	<li>As a control, plate 1/10th of the transformation mixture on one EMM2 plate (150 mm) with required supplements, but lacking 4-NQO, to calculate the total number of library clones obtained after transformation of the library, and screen for isolation of the suppressor clones.</li>
</ol>
2.&#160;Test and validate the rescue/suppression of the phenotype associated with the query mutant strain by the putative suppressor
NOTE: Stress spot assays were carried out as described below to test and validate the rescue/suppression of the ell1 deletion-associated 4-NQO stress sensitivity by the putative suppressor(s).
<ol>
	<li>Add 100-200 &#181;L sterile water in each of the different wells of a sterile 96-well microtiter plate.</li>
	<li>Pick a small amount of inoculum from each of the different colonies obtained after cDNA library transformation on the plate containing 4-NQO using a sterile toothpick or a 20-200 &#956;L micropipette tip. Add the inoculum from each of the different colonies into separate independent wells of the microtiter plate containing 100-200 &#181;L of sterile water. Mix thoroughly.</li>
	<li>Spot 3 &#181;L of the cell suspension from each well onto EMM2 agar plates containing adenine (225 &#181;g/mL) and uracil (225 &#181;g/mL) but lacking leucine (the selectable auxotrophic marker present on the plasmid vector used for construction of the library used in this work). Add appropriate concentrations of 4-NQO to the plates as required.</li>
	<li>Incubate the plates at 32 &#176;C for 3-4 days to allow the cells to grow. Identify the colonies that show growth in the presence of different concentrations of 4-NQO as putative suppressors.</li>
	<li>To further validate the suppressors, grow the selected suppressors along with appropriate control strains overnight at 32 &#176;C with shaking (200-250 rpm) in EMM2 medium containing 225 &#181;g/mL each of adenine and uracil but lacking leucine.</li>
	<li>Next day, dilute the cells to an OD600 nm of 0.3 in fresh EMM2 medium and grow them at 32 &#176;C with shaking until mid-log phase (approximately OD600 nm of 0.6-0.8).</li>
	<li>Spot appropriate serial dilutions (1:10 or 1:5) of cultures on EMM2 plates with required supplements containing either 0.4 &#181;M 4-NQO or 0.01% MMS. For control, spot the strains on an EMM2 plate with required supplements but lacking any DNA-damaging agent.</li>
	<li>Incubate the plates at 32 &#176;C for 3-5 days to monitor growth. 
	NOTE: Suitable assays based on the phenotype associated with the query mutant strain should be used to test the rescue or suppression of the phenotype. For example, if suppressors of a cold-sensitive phenotype of a query mutant strain need to be identified, the assay for testing and validation of the suppressor clones would involve growing transformants at low temperature.</li>
	<li>Spot the mutant strains transformed with full-length gene of interest (Spell1 in this case) or empty vector as positive and negative controls, respectively, along with the library transformants.</li>
</ol>
3.&#160;Isolation of the plasmid from the S. pombe suppressor clones
NOTE: Plasmid isolation from S. pombe was carried out by following the protocol described in Fission yeast: a laboratory manual28 with a few modifications.
<ol>
	<li>Inoculate a single yeast colony in EMM2 medium containing 225 &#181;g/mL of adenine and uracil and grow the cells overnight at 32 &#176;C with shaking at 200-250 rpm. Next day, harvest 5 O.D. cells (i.e., 10 mL culture of O.D. 0.5) by centrifuging for 2 min at 4,000 &#215; g at room temperature.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 0.2 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris HCl (pH 8.0), and 1 mM Na2EDTA).</li>
	<li>Add 0.2 mL of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3 g of acid-washed glass beads to the microcentrifuge tube. Vortex the microcentrifuge tube for 2 min at 4 &#176;C and incubate on ice for 1 min. Repeat this step 6x.</li>
	<li>Centrifuge the tube at 10,000 &#215; g for 15 min at room temperature. Transfer the upper aqueous layer to a fresh microcentrifuge tube and add 200 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1).</li>
	<li>Centrifuge the tube at 10,000 &#215; g for 10 min. Transfer the upper aqueous layer to a fresh tube and add two volumes of 100% ethanol (~400 &#181;L) and 1/10 volume of sodium acetate (3 M, pH 5.8) (~20 &#181;L) to the tube. Incubate the microcentrifuge tube -70 &#176;C for 1 h.</li>
	<li>Precipitate the DNA by centrifugation at 10,000 &#215; g for 15 min at 4 &#176;C and remove the supernatant. Wash the DNA pellet with 70% ethanol (~500 &#181;L) and keep it at room temperature to air-dry.</li>
	<li>Resuspend the DNA in 20 &#181;L of sterile water. Use 2-5 &#181;L of plasmid DNA for transformation of competent E. coli cells. 
	NOTE: Plasmid can also be isolated from yeast cells using any of the commercially available yeast plasmid isolation kits.</li>
</ol>
4.&#160;Identification of the gene encoded by the suppressor clone
<ol>
	<li>Transform the isolated yeast plasmids into E. coli Top10 strain [F-mcrA (mrr-hsdRMS-mcrBC) 80LacZM15 lacX74 recA1 ara139 (ara-leu)7697 galUgalKrpsL (StrR) endA1 nupG] using the standard protocol29 and spread the cells on LB (Luria Broth) plates with required antibiotics.</li>
	<li>Isolate the plasmid(s) from E. coli transformants using the standard alkaline lysis protocol29, and follow the appropriate combinations of restriction enzymes to check for the release of the insert DNA fragment by restriction digestion. 
	NOTE: Suppressor plasmid 84 was digested with BamHI restriction enzyme, and Suppressor plasmid 104 was digested with PstI/BamHI restriction enzymes.</li>
	<li>Retransform the plasmids showing insert release after restriction digestion in the ell1&#916;&#160;strain to check for their ability to rescue the genotoxic stress-sensitive phenotype of the ell1&#916;&#160;strain.</li>
	<li>Select the plasmid clones showing suppression of the genotoxic stress sensitivity and sequence the insert DNA fragment present in these plasmid clones using vector-specific forward and reverse primers. 
	NOTE: In this study, the adh1 promoter-specific universal forward primer (5&#39;CATTGGTCTTCCGCTCCG 3&#39;) was used30.</li>
	<li>To identify the gene responsible for the suppression, align the sequence obtained using NCBI nucleotide blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&#38;BLAST_SPEC=GeoBlast&#38;PAGE_TYPE=BlastSearch) or Pombase sequence alignment tool (https://fungi.ensembl.org/Schizosaccharomyces_pombe/Tools/Blast?db=core). Select the sequence showing maximum alignment and identify it as the gene for the multicopy suppressor plasmid.</li>
</ol>

The authors have no conflicts of interest.

Yeasts have been widely used to investigate the basic biological processes and pathways that are evolutionarily conserved across eukaryotic organisms. The availability of genetic and genomic tools along with their amenability to various biochemical, genetic, and molecular procedures make yeasts an excellent model organism for genetic research34,35,36. Over the years, various genetic screens have been designed by yeast researcher...

Networks of genetic interactions provide functional information about genes and delineate pathways and biological processes that these genes may be involved in in vivo. In addition, they may also provide insights into how different genes interact with one another, resulting in a specific phenotype1,2,3. Over the years, a variety of genetic screens have been designed by researchers to answer fundamental biological questions and study human diseases. Screens for the identification of genetic interactions can be performed in multiple ways. Genetic interactions identified in different genetic screens can represent distinct mechanistic relationships between genes. Furthermore, studies have revealed that a common set of genetic interactions are shared by genes that encode proteins belonging to the same pathway or complex4,5. Thus, genetic interaction networks can be used to establish the functional organization in a cell, wherein genes sharing the most similar profiles belong to the same complex or pathway, those genes sharing somewhat less similar profiles belong to the same biological process, and those genes exhibiting overlapping but more diverse profiles reflect members belonging to the same cellular compartment6.
Genetic interaction screens based on dosage suppression (&#39;high-copy or multicopy suppression&#39;) are one of the commonly used approaches. These screens can be performed by transforming a query mutant strain with a high-copy-number genomic or cDNA library, followed by suitable assays/selection techniques to identify suppressing or enhancing genetic interactions7,8,9. To ensure a comprehensive genome-wide coverage, these screens have also been carried out by overexpressing a specific gene of interest in a collection of genome-wide loss-of-function mutant or by overexpressing a high-copy-number plasmid-encoded genomic or cDNA library in a loss-of-function query mutant9,10,11,12,13,14,15. The multicopy strategy could also work using a dominant/overexpression approach using a regulatable promoter.
The main advantages of using suppressor-based screens are that suppression of a preexisting phenotype in a mutant strain by another gene establishes a genetic relationship among these two gene products that may not have been demonstrated using other approaches. Second, it has been observed that the presence of a preexisting mutation sensitizes a particular pathway, allowing additional components of that pathway to be identified by the isolation of suppressors, which may have not been identified by more direct genetic selections. Moreover, this screen can be used to identify suppressors that have different mechanisms of suppression16. Suppressor interactions usually occur between genes that are functionally related and can be used to elucidate hierarchies in pathways. The exact underlying mechanism of suppression may differ based on several factors, including the type of query mutant used in the screen, experimental conditions, and the level of gene expression. One of the common dosage suppression mechanisms involves genes encoding products that function together in the same complex or in parallel in the same cellular/biological process. The results of such screens in simpler model organisms such as yeast can be extended to higher eukaryotic organisms since most fundamental biological pathways and processes are conserved across evolution.
These genetic screens can also be modified in several ways to answer different biological questions. For example, orthologous genes from different organisms that can suppress the phenotype of the query mutant strain can be identified. It has also been used to delineate potential resistance mechanisms and determine protein targets of novel antibacterial17,18, antifungal19,20, antiparasitic21, and anticancer22 compounds. This screen has also been exploited to identify suppressors of the activity of pharmaceutical drugs whose mechanism of action is not known. Thus, in principle, these multicopy suppressor screens can be optimized and used in a variety of applications in different organisms. Although most of the genetic screens employed by yeast researchers have been initially developed in S. cerevisiae, S. pombe has emerged as a complementary model system for&#160;carrying out various genetic screens and assays23. Moreover, genomic organization and biological&#160;processes in S. pombe, such as occurrence of introns in more genes, complexity of origins of DNA replication, centromere structure, organization of the cell cycle and presence of the RNAi&#160;machinery, show greater resemblance between S. pombe and higher eukaryotes23,24, underscoring&#160;the importance of designing and using genetic tools in S. pombe.
This paper describes a protocol for identifying genetic interactors based on &#39;dosage suppression&#39; of a loss-of-function mutant phenotype in S. pombe. The basis of this protocol is that it is a rapid and efficient method to screen a cDNA library overexpressing wild-type genes either on a multicopy plasmid and/or from a strong promoter. This protocol has four main steps: transformation of the library into a query mutant strain, selection of plasmid clones that suppress the desired phenotype of the query mutant strain, retrieving the plasmid(s) from these suppressor clones, and identification of the gene responsible for the suppression of the phenotype. As is true for any method based on the selection and identification of cDNAs from a library, the success of the screen is dependent on using a high-quality and high-complexity library as the screen can retrieve only those cDNA clones that are present in the library.
Using this protocol, we have successfully identified two novel suppressors of the genotoxic stress-sensitive phenotype of the query S. pombe ell1 null mutant. The ELL (Eleven Nineteen Lysine Rich Leukemia) family of transcription elongation factors suppress transient pausing of RNA polymerase II on DNA templates in in vitro biochemical assays and are conserved across various organisms, from fission yeast to humans25. Earlier work has provided evidence that an S. pombe ell1 null mutant shows genotoxic stress sensitivity in the presence of 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS)26. Therefore, we transformed a S. pombe plasmid-encoded multicopy cDNA library into the query S. pombe ell1 null mutant and identified two putative clones that exhibited the ability to suppress the genotoxic stress sensitivity of the S. pombe ell1 null mutant in the presence of 4-NQO, a compound that induces DNA lesions. Subsequent sequencing of the insert present in the plasmid clones identified that the genes encoding rax2+ and osh6+ were responsible for suppressing the genotoxic stress sensitivity of ell1 null mutant when overexpressed in the ell1 null mutant.

<table><tbody><tr><td>4-NQO</td><td>Sigma</td><td>N8141</td><td></td></tr><tr><td>Acetic Acid, glacial</td><td>Sigma</td><td>1371301000</td><td></td></tr><tr><td>Adenine Sulphate</td><td>Himedia</td><td>GRM033</td><td></td></tr><tr><td>Agar</td><td>Himedia</td><td>GRM026</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004L</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Himedia</td><td>MB054</td><td></td></tr><tr><td>BamHI</td><td>Fermentas</td><td>ER0051</td><td></td></tr><tr><td>Biotin</td><td>Himedia</td><td>RM095</td><td></td></tr><tr><td>Boric Acid</td><td>Himedia</td><td>MB007</td><td></td></tr><tr><td>Calcium Chloride&amp;nbsp;</td><td>Sigma</td><td>C4901</td><td></td></tr><tr><td>Chloroform:Isoamyl alcohol 24:1</td><td>Sigma</td><td>C0549</td><td></td></tr><tr><td>Citric Acid</td><td>Himedia</td><td>RM1023</td><td></td></tr><tr><td>Disodium hydrogen phospahte anhydrous</td><td>Himedia</td><td>GRM3960</td><td></td></tr><tr><td>single stranded DNA from Salmon testes</td><td>Sigma</td><td>D7656</td><td></td></tr><tr><td>EDTA disodium</td><td>Sigma</td><td>324503</td><td></td></tr><tr><td>Ferric Chloride Hexahydrate</td><td>Himedia</td><td>RM6353</td><td></td></tr><tr><td>Glucose</td><td>Amresco</td><td>188</td><td></td></tr><tr><td>Ionositol</td><td>Himedia</td><td>GRM102</td><td></td></tr><tr><td>Isopropanol</td><td>Qualigen</td><td>Q26897</td><td></td></tr><tr><td>Leucine</td><td>Himedia</td><td>GRM054</td><td></td></tr><tr><td>Lithium Acetatae</td><td>Sigma</td><td>517992</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Himedia</td><td>MB040</td><td></td></tr><tr><td>Molybdic Acid</td><td>Himedia</td><td>RM690</td><td></td></tr><tr><td>Nicotinic Acid</td><td>Himedia</td><td>CMS177</td><td></td></tr><tr><td>PEG, MW 4000</td><td>Sigma</td><td>81240</td><td></td></tr><tr><td>Pentothinic Acid</td><td>Himedia</td><td>TC159</td><td></td></tr><tr><td>Phenol</td><td>Himedia</td><td>MB082</td><td></td></tr><tr><td>Plasmid Extraction Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Potassium hydrogen Pthallate</td><td>Merc</td><td>DDD7D670815</td><td></td></tr><tr><td>Potassium iodide</td><td>Himedia</td><td>RM1086</td><td></td></tr><tr><td>RNAse</td><td>Fermentas</td><td>EN0531</td><td></td></tr><tr><td>SDS</td><td>Himedia</td><td>GRM205</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Himedia</td><td>GRM1183</td><td></td></tr><tr><td>Sodium Sulphate</td><td>Himedia</td><td>RM1037</td><td></td></tr><tr><td>Tris free Base</td><td>Himedia</td><td>MB209</td><td></td></tr><tr><td>Uracil</td><td>Himedia</td><td>GRM264</td><td></td></tr><tr><td>Yeast Extract Powder</td><td>Himedia</td><td>RM668</td><td></td></tr><tr><td>Zinc Sulphate Heptahydrate</td><td>Merc</td><td>DJ9D692580</td><td></td></tr></tbody></table>

genetic screen for identification of multicopy suppressors in schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Screening for multicopy suppressor(s) of ell1 deletion-associated genotoxic stress sensitivity in S. pombe 
We performed the genetic screen using the protocol described above to identify multicopy suppressors of the loss-of-function phenotype of the query ell1 deletion mutant strain. The growth-related sensitivity of the ell1 deletion strain observed in the presence of the 4-NQO genotoxic agent was adopted as the ...

Watch this Scientific Journal Video about Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe at JoVE.com

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

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1.7K Views.  University School of Biotechnology, G.G.S. Indraprastha University. This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Video: Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Genetics

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

Cancer Research

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Summary

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Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Competitive Genomic Screens of Barcoded Yeast Libraries

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

High-throughput Screening for Protein-based Inheritance in S. cerevisiae