James T. Arnone would like to acknowledge the support of the students in the Recombinant DNA Technologies course in 2017 and 2018 at William Paterson University who were involved in this project from its inception, but whose efforts did not cross the threshold for authorship: Christopher Andino, Juan Botero, Josephine Bozan, Brenda Calalpa, Brenda Cubas, Headtlove Essel Dadzie, Irvin Gamarra, Preciousgift Isibor, Wayne Ko, Nelson Mejia, Hector Mottola, Rabya Naz, Abdullah Odeh, Pearl Paguntalan, Daniel Raza&#8217;e, Gabriella Rector, Aida Shono, and Matthew So. You are great scientists and I miss you all!
The authors would like to acknowledge the invaluable support of Instruction and Research Technology at William Paterson University for their help: Greg Mattison, Peter Cannarozzi, Rob Meyer, Dante Portella, and Henry Heinitsh. The authors would also like to acknowledge the Office of the Provost for ART support, the Office of the Dean and the Center for Research in the College of Science and Health their support of this work, and the Department of Biology for supporting this project.

1. Identify potential genetic interactions for screening
<ol>
	<li>Identify the genetic background(s) for characterization, that results in an abnormally shorted chronological life span (CLS) in Saccharomyces cerevisiae using the Saccharomyces Genome Database (the SGD, https://www.yeastgenome.org29,30), which compiles known phenotypic information for this organism.
	<ol>
		<li>Select the Function tab from the options on the top of the webpage.</li>
		<li>Select Phenotype followed by selecting Browse all Phenotypes.</li>
		<li>From the Yeast Phenotype Ontology options scroll to the Development subheading and select Chronological Lifespan, found under the Lifespan subheading.</li>
		<li>Select the qualifier for decreased, which allows for the identification of genes that exhibit a phenotype that results in a decreased chronological lifespan phenotype when deleted. For this proof-of-method atg1&#916; was selected, which results in a short-lived CLS phenotype and is disrupted for autophagy26.</li>
	</ol>
	</li>
	<li>Identify target gene(s) to screen for genetic interactions that may suppress the phenotype, based on reported or predicted ontology attributes, of the mutant identified in part 1.2. Repeat the phenotype search as found in steps 1.1.1-1.1.4 above, querying for genes that result in a longer CLS when overexpressed in a wild-type background. SIR2 was selected based on the reported CLS phenotype and reported interactions with autophagy31,32.</li>
</ol>
2. Prepare reagents
NOTE: Unless otherwise specified autoclave each solution at 121 &#730;C for 20 min to sterilize prior to use.
<ol>
	<li>YPAD liquid media: Add 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose), and 40 mg adenine (as adenine sulfate dehydrate) per liter of double distilled water. Mix well with a magnetic stirrer.</li>
	<li>LB liquid media: Add Tryptone (10 g), Yeast extract (5 g), and Sodium Chloride (10 g) per liter of double distilled water. Mix well with a magnetic stirrer.</li>
	<li>Prepare 1000x (100 mg/mL) ampicillin stock, in double distilled water. Mix well and filter sterilize.</li>
	<li>Synthetic complete &#8211; Leucine (SC-LEU) liquid media: Add 1.7 g of Yeast nitrogen base w/o amino acids, 2% Glucose, 1.92 g of SC-LEU Dropout mix, 5 g of Ammonium sulfate per liter of double distilled water. Mix well with a magnetic stirrer.</li>
	<li>TE buffer: Mix Tris (10 mM final concentration), EDTA (1 mM final concentration) in the solution with double distilled water. Mix well with a magnetic stirrer.</li>
	<li>Prepare 50% PEG 3350 in solution with double distilled water. Mix well with a magnetic stirrer.</li>
	<li>Prepare 1 M and 100 mM Lithium Acetate solution with double distilled water. Mix well with a magnetic stirrer. 
	NOTE: To make solid agar plates add 20 g of agar (per liter with double distilled water) to the media prepared in 2.1 and 2.2 above prior to autoclaving. If preparing ampicillin plates add 1mL of the ampicillin to the media in 2.2 above after it has cooled to roughly 60 &#730;C. Pour into sterile plates and allow to set for 48&#8211;72 h prior to use. Store plates at 4 &#730;C for longer storage.</li>
</ol>
3. Design the cloning strategy to clone SIR2 into the pRS315 vector
<ol>
	<li>Design PCR primers to amplify the SIR2 gene for cloning into the pRS315 vector.
	<ol>
		<li>Design primers manually to have 21&#8211;22 nucleotide complementarity to the intergenic regions upstream and downstream of the SIR2. Ensure that the entire gene, along with the untranslated regions of the mRNA is cloned by mapping those features from the available datasets33,34.</li>
		<li>Ensure that the PCR primer design results in forward and reverse primers that have a melting temperature (Tm) above 53 &#176;C and below 60 &#176;C. 
		NOTE: Ideally, both primers should have a Tm as close to each other as sequence will allow, with an approximate GC content of between 40&#8211;50%, making sure to avoid dinucleotide repeats and balancing GC and AT distribution throughout the sequence.</li>
		<li>After the design of the PCR primers that will allow for the generation of the amplicon for cloning, add restriction enzyme digestion (R.E.D.) target sites to the 5&#8217; end of each primer that are compatible to the plasmid-cloning vector. In this method, a HindIII restriction enzyme digestion site (5&#8217;-AAGCTT-3&#8217;) is added to the upstream, forward primer and a SacII restriction enzyme digestion site (5&#8217;-CCGCGG-3&#8217;) is added to the downstream, reverse primer. 
		NOTE: The use of the SacII and HindIII sites requires that the consensus cut site for each endonuclease is not present in the target gene. If either enzyme targets within the target gene, alternative restriction enzymes should be chosen. There are many that are compatible with the polylinker region on the pRS315 vector.</li>
		<li>Lastly, add a four nucleotide (5&#8217;-NNNN-3&#8217;) sequence overhang to the 5&#8217; end of each primer to allow the restriction enzyme to bind and digest the amplicon. Once the primers have been designed, have the oligonucleotides commercially synthesized for use cloning the SIR2 gene.</li>
		<li>Resuspension of the PCR primers: Centrifuge the PCR primers using a tabletop microfuge at maximum speed for 4 min. Add TE solution to make a stock concentration of 100 &#181;M. Store the stock concentration at -20 &#176;C and dilute 1/10 for use in PCR applications. 
		NOTE: To make a 100 &#956;M stock, dissolve the primers in a volume of sterile TE buffer that is 10x the amount of nmoles in the primer tube, using microliters of TE. For example, if the tube contains 15.6 nmoles of primer, add 156 &#956;L of TE buffer.</li>
	</ol>
	</li>
	<li>Isolate wild-type yeast gDNA for PCR amplification of the SIR2 cloning construct. 
	NOTE: Several high-quality options are commercially available for isolating yeast gDNA. Utilization of a kit that includes the digestion of the fungal cell wall with zymolyase results in better quality gDNA (higher yield, less impurities). The protocol below consistently returns high concentration and purity. Details of a kit we recommend can be found in the Table of Materials.
	<ol>
		<li>Grow 5 mL culture of wild-type yeast for 48-72 h to post-log phase in enriched media, such as YPAD. Pellet the yeast cells at &#62;800 x g for 3 min at room temperature, remove the growth media, and resuspend in 120 &#956;L of zymolyase digestion buffer supplemented with 5 &#956;L of zymolyase (2 units enzyme/&#956;L). Mix the sample by vortexing and incubate at 37 &#176;C for 40 min.</li>
		<li>Add 120 &#956;L of a chaotropic lysis buffer (e.g., guanidinium chloride), 250 &#956;L of chloroform, and vortex the sample for 60 s.</li>
		<li>Centrifuge at &#62;8,000 x g for 2 min and transfer the supernatant into a purification column in a sterile collection tube.</li>
		<li>Centrifuge at &#62;8,000 x g for 60 s and discard the flow through. The gDNA will be bound to the column matrix.</li>
		<li>Wash the column twice with 300 &#956;L of an ethanol-based wash buffer, repeating the centrifugation step from above (3.2.4). Discard the flow through after each spin. Transfer the column into a 1.5 mL microfuge tube, add 60 &#956;L of TE buffer, and incubate at room temperature for 60 s. Flash spin the sample for 30 s to elute the DNA. 
		NOTE: Determine the concentration of the DNA in the sample (Absorbance at 260nm) and the quality (Absorbance 260nm/280nm). A typical yield will be 100&#8211;200 ng/ &#956;L of gDNA with absorbance ratio at 260nm/280nm as close to 1.8 as possible.</li>
	</ol>
	</li>
	<li>Amplify and isolate the pRS315 plasmid vector for cloning. 
	NOTE: Several high-quality options are commercially available for the purification of plasmid vectors. A silica-based column chemistry is recommended for this step. The changes noted below have led to the highest concentration and purity. Details are found in the Table of Materials.
	<ol>
		<li>Grow a 5 mL of culture of E. coli containing the pRS315 vector overnight in LB+ ampicillin (80 &#956;g/mL) media. Pellet the culture by centrifugation at &#62;8,000 x g for 2 min at RT (15&#8211;25 &#176;C).</li>
		<li>Re-suspend pelleted bacterial cells in 250 &#956;L of TE buffer with RNase A (100 &#956;g/mL) and transfer to a microcentrifuge tube. Ensure that no clumps of cells remain.</li>
		<li>Add 250 &#956;L of lysis buffer and mix by inverting the tube 6&#8211;8 times. Incubate for 5 min at room temperature. Do not allow lysis to proceed for more than 5 min &#8211; a little less is preferable.</li>
		<li>Add 350 &#956;L of neutralization buffer and mix immediately and thoroughly by inverting the tube 10 times. Centrifuge for 10 min at &#62;8,000 x g.</li>
		<li>Carefully transfer the supernatant from above to a silica spin column by pipetting. Centrifuge for 30 s and discard the flow-through.</li>
		<li>Add 500 &#956;L of a high salt wash buffer and centrifuge as in step 3.3.5. Discard the flow-through. Wash the DNA binding spin column by adding 750 &#956;L of an ethanol-based wash buffer, to remove residual salts, and centrifuge as in step 3.3.5.</li>
		<li>Discard the flow through and centrifuge for an additional 2 min at &#62;8,000 x g to remove residual wash buffer. Place the spin column in a clean, labeled 1.5 mL microcentrifuge tube. To elute DNA, add 20 &#956;L of TE buffer to the center of the spin column, incubate for 1 min at room temperature, and centrifuge for 1 min at &#62;8,000 x g.</li>
		<li>Use a spectrophotometer to determine the quantity (Absorbance at 260nm) and the quality (Absorbance 260 nm/280 nm) of DNA. A typical yield will be 1&#8211;2 &#956;g/&#181;L.</li>
	</ol>
	</li>
	<li>PCR amplification of the candidate gene, SIR2, from wild-type genomic DNA
	<ol>
		<li>To produce an amplicon that is suitable for cloning, utilize a high-fidelity (HF) PCR polymerase to avoid the unintentional generation of mutations into the sequence being amplified. 
		NOTE: Many different high-fidelity PCR options are commercially available. To facilitate the optimization of the PCR reaction conditions, use two-buffer combination: one that is a standard HF buffer and one optimized for high GC and complex amplicons. Details can be found in the Table of Materials.</li>
		<li>PCR amplify the SIR2 construct for cloning as described in Table 1. 
		NOTE: To maximize success in the cloning steps, multiple identical 50 &#956;L can be set up and concentrated by a PCR column clean up step. Be sure to set up one no gDNA template control reaction (negative control).</li>
		<li>Set up the PCR cycling conditions as described in Table 2. 
		NOTE: Different primer pairs vary on their annealing temperature and different polymerases function at different speeds. Make sure to optimize the amplification conditions based upon the enzyme selected and the specifications of the primer combination as designed.</li>
		<li>Verify the success of the PCR reaction by visualizing the PCR reaction, which would produce an approximately 2.5 kb of DNA fragment, on a 1.0% TAE-agarose gel (with 0.5 &#956;g/mL ethidium bromide for visualization).</li>
	</ol>
	</li>
	<li>Digestion and ligation of the candidate gene, SIR2, into the pRS315 plasmid vector.
	<ol>
		<li>Perform restriction digestion of the vector and the insert: 625 ng DNA (either the vector or insert), q.s. water to bring the final reaction volume to 50 &#956;L, 5 &#956;L of buffer, 1 &#956;L SacII, and 1 &#956;L HindIII. Incubate the restriction digestions at 37 &#176;C for 3 h, followed by 80 &#176;C for 20 min to heat inactivate the enzymes. Digests can be stored at 4 &#176;C prior to proceeding to the next step.</li>
		<li>Set up a 15 &#956;L ligation reaction to create the desired plasmid: 6 &#956;L of sterile water, 2 &#956;L digested vector (50 ng DNA), 4 &#956;L digested insert (100 ng DNA), 2 &#956;L T4 reaction buffer, and 1 &#956;L of T4 DNA Ligase. Incubate the ligation reactions overnight at 16 &#176;C, followed by 80 &#176;C for 20 min to heat inactivate the enzyme. 
		NOTE: Set up a no insert control, substituting an additional 4 &#956;L of sterile water (10 &#956;L total) in lieu of the insert.</li>
		<li>Transform the ligation reactions into E. coli. 
		NOTE: There are many options available for competent cells that are available. This protocol uses chemically competent cells that are stored at -80 &#176;C prior to use.
		<ol>
			<li>Thaw a 50 &#956;L tube of frozen, competent E. coli cells on ice until just thawed and immediately add 15 &#956;L of the ligation reaction. Flick the tube several times. Immediately return the tubes to ice and incubate for 30 min.</li>
			<li>Heat-shock the cells for 20 s in a water bath at exactly 42 &#176;C, and immediately return the tubes to ice for a 2 min incubation. Add 450 &#956;L of room temperature recovery media (e.g., SOC or LB) to each transformation reaction and incubate for 60 min at 37 &#176;C with shaking.</li>
			<li>For each transformation reaction, make a 1:10 dilution of cells. Using sterile technique, plate 150 &#181;L of the undiluted cells and the 1:10 dilutions onto LB + (80 &#956;g/mL) ampicillin plates35. Incubate the plates at 37 &#176;C overnight.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Screen prospective transformants for the overexpression vector.
	<ol>
		<li>Using sterile technique, inoculate the potential transformants that grew into 5 mL LB + (80 &#956;g/mL) ampicillin and grow overnight. Following the procedure outlined in section 3.3.1&#8211;3.3.7 above, isolate the plasmids from every potential transformant and screen for successful integration of the insert by restriction digestion followed by gel electrophoresis on a 1.0% TAE-agarose gel (with 0.5 &#956;g/mL ethidium bromide for visualization).</li>
	</ol>
	</li>
</ol>
4. Transform the vector into atg1&#916; and wild type yeast strains
NOTE: This is performed using a modified lithium acetate transformation protocol36.
<ol>
	<li>Pellet 15 mL of wild type and atg1-null yeast cells grown overnight in YPAD media to early to mid-log phase (O.D. 600nm = 0.4-0.9) of growth for 3 min at &#62; 800 x g at room temperature.</li>
	<li>Decant the supernatant, re-suspend the cell pellet in 1 mL of sterile ddH2O and transfer the contents to a 1.7 mL microfuge tube. Pellet the cells for 3 min at &#62; 800 x g at room temperature.</li>
	<li>Remove the supernatant and re-suspend the cells in 250 &#956;L of 100 mM lithium acetate with gentle pipetting. Split the cells into separate microfuge tubes for each of the transformations that you will perform. Use 50 &#956;L of the cell-lithium acetate mix per transformation.</li>
	<li>Set up a transformation mix. To each of the transformations add: 240 &#956;L of 50% PEG3350, 36 &#956;L of 1.0 M Lithium acetate, and 5 &#956;L of Salmon sperm (or other carrier) DNA, boiled for 5 min and on ice. 
	NOTE: PEG is very viscous. Pipette and measure carefully. Mix by pipetting after the addition of each component prior to moving on.</li>
	<li>Add 5 &#956;L of the appropriate plasmid for each transformation performed. Vortex each tube to mix thoroughly. Incubate the samples at 30 &#176;C for 45 min. Heat shock samples at 42 &#176;C for 10 min.</li>
	<li>Pellet cells for 3 min at &#62; 800 x g at room temperature, carefully remove the transformation mix, and re-suspend samples in 300 &#956;L of sterile ddH2O. Pellet cells by repeating the spin above, carefully remove water, and re-suspend your samples in 200 &#956;L of sterile ddH2O.</li>
	<li>Set up 1/10 and 1/100 dilutions for each transformed strain of yeast.</li>
	<li>Using sterile technique plate 150 &#956;L from each sample onto SC-leucine plates to select for the plasmids. Spread the cells uniformly and evenly and allow the plate to dry before inverting and incubating at 30 &#176;C to grow for 48&#8211;72 h. 
	NOTE: Once the appropriate strain is generated, it may be stored long term in 25% glycerol at -80 &#176;C. The quantification of copy number present can be determined by several methodologies, including qPCR, RNA-FISH, or another appropriate measure37,38.</li>
</ol>
5. Determine the chronological life span to test for shortened CLS phenotype suppression
<ol>
	<li>Test the effect of overexpression of the putative suppressor on CLS in the short-lived yeast, atg1&#916; mutant, by determining the number of colony forming units (CFUs) that remain as a function of time39. 
	NOTE: It is necessary to set up this portion of the experiment with the appropriate controls. A typical experiment will compare a wild type (WT) strain of yeast with the empty vector, WT with the suppressor vector, the deletion mutant with the empty vector, and the deletion mutant with the suppressor vector.
	<ol>
		<li>Take a single colony of the strain to study and inoculate it into SC-LEU media. Grow the culture at 30 &#176;C for 72 h, with shaking.</li>
		<li>Using a hemocytometer, determine the concentration of cells that are present in the culture40.</li>
		<li>Dilute an aliquot of the culture, so that the result is a uniform number of cells in a 150 &#181;L volume of sterile water. Plate the culture using sterile technique onto SC-LEU plates and grow at 30 &#176;C for 72 h. These plates are the day three time-point and the experiment will be normalized to this time-point as 100% viability39. 
		NOTE: The number of cells should be 200&#8211;500, sufficiently large for the analysis and a manageable number for counting. In this study, we used 200 cells for our plating quantity.</li>
		<li>Continue to incubate the yeast cultures at 30 &#176;C, taking regular aliquots and plating as outlined in 5.1.3. Continue this process until the strains are no longer viable, then compile and analyze the results. 
		NOTE: The complete list of strains used in this study are found in Table 3.</li>
	</ol>
	</li>
</ol>

The authors declare that there is no conflict of interest.

Unravelling the genetics of aging is a difficult challenge, with many opportunities for further study that can potentially yield significant insights into the complex interactions that exist. There are many methods that allow for the rapid generation of loss-of-function mutants for the study of null strains of yeast45,46. This method presents a straightforward approach to identify and clone genes onto the pRS315 vector for overexpression suppressor studies. One a...

Aging is the time-dependent loss of integrity in myriad biological processes that ultimately increases the probability of organismal death. Aging is nearly inevitable for all species. On a cellular level there are several well characterized hallmarks that are associated with aging, including: genomic instability, epigenetic alterations, loss-of-proteostasis, mitochondrial dysfunction, deregulated nutrient sensing, cellular senescence, and telomere attrition1,2. In single celled organisms, such as yeasts, this leads to a reduction in replicative potential and chronological life span3,4. These cellular changes manifest in more complex organisms, like humans, as pathologies that include cancers, heart failure, neurodegeneration, diabetes, and osteoporosis5,6,7. Despite the many complexities that characterize the process of aging, there is conservation of these molecular hallmarks underlying this process across widely divergent organisms8,9,10. Identification of alterations to these pathways during aging led to the realization that they can be manipulated via lifestyle changes &#8211; dietary restriction is shown to substantially extend lifespan in many organisms11. These pathways converge and intersect with each other and many other pathways, in complex ways. Elucidation and characterization of these interactions offers potential for therapeutic interventions to prolong lifespan and healthspan12,13,14.
The conservation of the molecular underpinnings of aging allows for functional dissection of genetic interactions underlying the process through the use of simpler model organisms &#8211; including in the budding yeast, Saccharomyces cerevisiae15,16. There are two established types of aging modeled by budding yeast: chronological aging (the chronological lifespan, CLS) and replicative aging (the replicative lifespan, RLS)17. Chronological aging measures the amount of time that a cell can survive in a non-dividing state. This is analogous to the aging that is seen in cells that spend the majority of their life in G0, such as neurons4. Alternatively, replicative lifespan is the number of times that a cell can divide before exhaustion and is a model for mitotically active cell types (e.g., the number of daughter cells that a cell can have)18.
The overall goal of this method is to present a protocol that allows for the functional dissection of the genetics of aging using S. cerevisiae. While there have been many excellent studies performed by many researchers that have led to our current understanding, there remain many opportunities available for budding researchers to contribute to the aging field from early in their academic career. We present a clear methodology that will allow researchers to further advance the field of aging. This protocol is designed to be accessible for all researchers regardless of the stage in their academic career by providing the tools necessary to formulate and test their own novel hypotheses. The advantage&#160;of our approach is that this is a cost effective method readily accessible to all researchers regardless of institution &#8211; and does not require expensive, specialized equipment necessary for some protocols19. There are several different ways to design this type of screen, the approach outlined in this work is particularly amenable to screening null mutants of non-essential genes that exhibit a severe reduction in the chronological lifespan compared to an isogenic wild-type strain of yeast.
As our proof of principle, we clone SIR2, a lysine deacetylase reported as exhibiting both an extended and a shortened CLS when overexpressed. SIR2 overexpression was recently found to increase CLS in winemaking yeasts; however, several groups have reported no link between SIR2 and CLS extension, leaving its role under characterized20,21,22. Due to these conflicting reports in the literature, we selected this gene to add independent research to help clarify the role of SIR2 in chronological aging, if any. Additionally, increasing the copy number of a SIR2 homologue extends lifespan in a nematode worm model system23.
Autophagy is an intracellular degradation system to deliver cytosolic products, such as proteins and organelles, to the lysosome24. Autophagy is intimately linked to longevity through its role in degrading damaged proteins and organelles to maintain cellular homeostasis25. Induction of autophagy depends on orchestrating the expression of many genes, and the deletion of the ATG1 gene results in an abnormally short CLS in budding yeast26. ATG1 codes for a protein serine/threonine kinase that is required for vesicle formation in autophagy and the cytoplasm-to-vacuole (the fungal lysosomal equivalent) pathway27,28. Here, we present our method for an increased copy number screen, testing the effect of increased SIR2 copy on the CLS in a wild type and an atg1-null background. This method is particularly amenable to junior researchers and research groups at primarily undergraduate institutions, many of which serve communities underrepresented in the sciences and have limited resources.

<table>
	<tbody>
		<tr>
			<td>Fungal/Bacterial DNA kit</td>
			<td>Zymo Research</td>
			<td>D6005</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIIIHF enzyme</td>
			<td>New England Biolabs</td>
			<td>R3104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid miniprep kit</td>
			<td>Qiagen</td>
			<td>12123</td>
			<td></td>
		</tr>
		<tr>
			<td>SacII enzyme</td>
			<td>New England Biolabs</td>
			<td>R0157S</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon sperm DNA</td>
			<td>Thermofisher</td>
			<td>AM9680</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

As there are conflicting reports on the role of SIR2 during aging, we chose this gene for study as a potential suppressor of the atg1&#916; mutant&#8217;s shorten CLS phenotype26. The role of SIR2 is somewhat controversial, with conflicting reports on its role in extending CLS, however it has been clearly linked to increased CLS in at least one yeast background, with a role in both autophagy and mitophagy22,31<su...

Watch this Scientific Journal Video about A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae at JoVE.com

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.

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 ...

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6.2K Views.  William Paterson University. 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.

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

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 ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

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 ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...