We would like to thank the Wellcome Trust Sanger Institute for the gifts of piggyBac transposon and piggyBac transposase. This work was supported by the Butcher grant of Colorado and the NIH R01 AG041801 (W.S.C).

<ol>
	<li>Johnson, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+genes+in+the+nematode+Caenorhabditis+elegans+also+mediate+increased+resistance+to+stress+and+prevent+disease.">Longevity genes in the nematode Caenorhabditis elegans also mediate increased resistance to stress and prevent disease.</a> J Inherit Metab Dis. 25, 197-206 (2002).</li><li>Murakami, S., Salmon, A., Miller, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+stress+resistance+in+cells+from+long-lived+dwarf+mice.">Multiplex stress resistance in cells from long-lived dwarf mice.</a> Faseb J. 17, 1565-1566 (2003).</li><li>Perez, V. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+overexpression+of+major+antioxidant+enzymes+does+not+extend+the+lifespan+of+mice.">The overexpression of major antioxidant enzymes does not extend the lifespan of mice.</a> Aging Cell. 8, 73-75 (2009).</li><li>Martin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+mice+to+test+the+oxidative+damage+theory+of+aging.">Genetic engineering of mice to test the oxidative damage theory of aging.</a> Ann NY Acad Sci. 1055, 26-34 (2005).</li><li>Chick, W. S., Drechsel, D. A., Hammond, W., Patel, M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+mutant+phenotypes+from+ES+cells+to+adult+mice.">Transmission of mutant phenotypes from ES cells to adult mice.</a> Mamm Genome. 20, 734-740 (2009).</li><li>Ramirez-Solis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+microextraction:+a+method+to+screen+numerous+samples.">Genomic DNA microextraction: a method to screen numerous samples.</a> Anal Biochem. 201, 331-335 (1992).</li><li>Uren, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+splinkerette-PCR+method+for+the+isolation+and+sequencing+of+retroviral+insertion+sites.">A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.</a> Nat Protoc. 4, 789-798 (2009).</li><li>Wang, W., Bradley, A., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+piggyBac+transposon-based+genome-wide+library+of+insertionally+mutated+Blm-deficient+murine+ES+cells.">A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells.</a> Genome Res. 19, 667-673 (2009).</li><li>Chick, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+stress-resistance+mutations+in+the+mouse.">Screening for stress-resistance mutations in the mouse.</a> Front Genet. 5, 310 (2014).</li><li>Salmon, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+cell+lines+from+young+adult+mice+of+long-lived+mutant+strains+are+resistant+to+multiple+forms+of+stress.">Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress.</a> Am J Physiol Endocrinol Metab. 289, 23-29 (2005).</li><li>Skarnes, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+public+gene+trap+resource+for+mouse+functional+genomics.">A public gene trap resource for mouse functional genomics.</a> Nat Genet. 36, 543-544 (2015).</li><li>Baker, K. E., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense-mediated+mRNA+decay:+terminating+erroneous+gene+expression.">Nonsense-mediated mRNA decay: terminating erroneous gene expression.</a> Curr Opin Cell Biol. 16, 293-299 (2004).</li><li>Shigeoka, T., Kawaichi, M., Ishida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+nonsense-mediated+mRNA+decay+permits+unbiased+gene+trapping+in+mouse+embryonic+stem+cells.">Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells.</a> Nucleic Acids Res. 33, e20 (2004).</li><li>Symula, D. J., Zhu, Y., Schimenti, J. C., Rubin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+annotation+of+mouse+mutations+in+embryonic+stem+cells+by+use+of+expression+profiling.">Functional annotation of mouse mutations in embryonic stem cells by use of expression profiling.</a> Mamm Genome. 15, 1-13 (2004).</li></ol>

The authors have nothing to disclose.

Forward genetic analysis allows for an unbiased interrogation of the genome for genes responsible for a specific phenotype. This method is very powerful to uncover novel gene functions. It has been widely used in lower organisms but not in mammal, such as the mouse, mainly due to the extremely high cost associated with the infrastructure and logistics that would entail. Here, we moved the genetic screening process to the ES cell culture platform, greatly increasing the efficiency and throughput in generating mutants and identifying mutations. 
To effectively select for resistant mutants, a lethal dose of stressor and period of treatment needs to be established such that all of the wild-type cells would be killed. The higher the stringency of the selection, the fewer occurrence of false positive will result. Fibroblast feeders have a higher tolerance to oxidative stress than ES cells; the presence of feeders would rescue the nearby ES cells which would otherwise be killed by the stressor. Thus, omission of feeders during stress selection was very critical for an effective killing. In skin fibroblasts, serum deprivation was necessary to reveal the stress resistance phenotype in long lived mice 10. However, this application was not feasible in ES cells because they cannot survive without serum. As an alternative, we discovered that the use of heat inactivated serum at reduced level (7.5%) achieved a similar result.
In this report, we employed poly-A trap rather than a promoter trap because promoter trap will only trap actively transcribed genes, which is estimated to be 60% of the genome in ES cells, under normal conditions 11. In contrast, a poly-A trap would trap genes regardless of their transcriptional activities, in theory giving us the potential to screen for mutations in all genes in the genome. A major drawback of the conventional poly-A trap is the strong bias towards trapping the last exon, appeared to be mediated by an endogenous nonsense-mediated decay (NMD) mechanism 12. By incorporating the unbiased poly-A trap (UPA) strategy in the PB-UPA transposon to suppresses NMD 13, we were able to eliminate biased trapping.
One distinguishing advantage of using ES cells is that these pluripotent cells make direct derivation of mice possible, speeding up the transition to in vivo studies. For this to happen, the replica plating strategy is critical, preserving a subset of clones sheltered from manipulations that can diminish pluripotency, such as knocking out of Blm and exposure to stressors. We have shown that mutant ES cells recovered from these screens were capable of participating in forming the entire mouse and transmitting the mutations to the germline. Most importantly, consistent with previous studies, the stress resistance phenotype was also transmitted and maintained in the adult mouse. 
We and others showed that a variant phenotype observed in ES cells resulting from environmental induced lesion or deliberated engineered mutations was able to transmit to the differentiated progeny cells 4,5,9,14, and probably the whole mouse. This protocol describes the utilization of ES cells as surrogate cells to screen for stress resistance mutations in the mouse. In addition to screening for stress resistance genes, this method may have a broader implication to screen ES cells, identify targets, and to develop a variety of mouse models with a phenotype of interest.

Longevity has an intimate relationship with stress resistance. In general, long-lived species often demonstrate increased resistance toward multiple stressors, such as hydrogen peroxide, paraquat (PQ), UV, heat, and heavy metals 1,2. In contrast, increased sensitivity to stress tends to predict shortened life span and/or a more disease-prone phenotype. The anti-oxidant scavenging pathway has long been speculated to play a major role in conferring stress resistance to the animal. However, with few exceptions, studies from a variety of transgenic animals with manipulations in various anti-oxidant enzymes (e.g., SOD) indicate that increasing the level of oxidant scavenging enzymes does not increase life span or health span 3. These data suggest that the stress resistance trait consistently observed in long-lived animals is mediated by other cellular pathways yet to be uncovered.
We took an unbiased forward genetic approach to identify genes, which upon mutated, could confer a stress resistance phenotype in cultured embryonic stem (ES) cells. ES cells offer two major advantages in this study: (1) sophisticated genetic manipulations are available to modify the genome of ES cells; and (2) any stress resistant ES cells recovered from the screen can be directly used for mouse production, allowing rapid translation into whole animal studies to measure life span and health span.
In this report, we described the use of C9 ES cell line, in which the Blm alleles were under control by a tetracycline responsive element. Treatment of doxycycline (dox) transiently turned off the expression of Blm leading to increased incident of sister chromatid exchange. This short-term Blm knock-out allowed for the generation of homozygous mutations within the heterozygote population so that recessive mutations for stress resistance could be captured in the screening process. We also described the use of piggyBac (PB) transposon as the mutagen to randomly insert a poly-A trap cassette (PB-UPA) to mutate genes in the genome. Cells with disruption of a gene by the poly-A trap became G418 resistant and could be recovered so that a collection of gene-trap mutants (gene-trap library) could be made, and subsequently screened for mutant clones that were stress resistant.
Stress resistant clones recovered from the selection could be characterized rather rapidly by molecular techniques in regard of the number of insertions (qPCR), the site of insertion (splinkerette PCR), the identity of the disrupted gene (BLAST), and its expression level (RT-qPCR). The PB insertion could be remobilized by transient expression of mPB transposase in the clone to restore the wild-type DNA sequence and thus test for the loss of stress resistance. These are powerful ways to confirm causality of the mutation, which should be done prior to expensive mouse production. Previous studies showed that cells exposed to stressors lost their pluripotency 4,5. Thus, in this protocol, the preservation of a replica set of mutant cells, which would not be treated with stressors is critical for successful mouse production.
Our lab has engineered the C9 ES cell line and the PB-UPA vector, both are available to other investigators upon request. The protocol reported here will start by the generation of de novo library of gene-trapped ES cells with PB-UPA (Figure 1A), followed by replica plating and stress selection to isolate stress resistant clones (Figure&#160;1B). We demonstrated the selection with paraquat, a potent free radical generator inside cells. Virtually, any cytotoxic compound or toxin, for instance, ER stressors (e.g., thapsigargin and tunicamycin), neuronal oxidant (e.g., MPP+, 6-hydroxy dopamine, and rotenone), heat, and heavy metals (e.g., Cd, Se), could be adapted to the method to select for respective resistant mutants.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Vector&lt;/strong&gt;</td></tr><tr><td>PB-UPA</td><td></td><td></td><td></td></tr><tr><td>mPBase</td><td></td><td></td><td></td></tr><tr><td>mPBase&lt;sup&gt;Puro&lt;/sup&gt;</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td></tr><tr><td>500-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>250-ml Stericup filters</td><td>EMD Millipore</td><td>SCGPU02RE</td><td></td></tr><tr><td>50-ml Steriflip-GV filters</td><td>EMD Millipore</td><td>SE1M179M6</td><td></td></tr><tr><td>KO DMEM</td><td>Life Technologies</td><td>10829-018</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>FBS</td><td>Tissue Culture Biologicals</td><td>104</td><td></td></tr><tr><td>Heat Inactivated FBS</td><td>Sigma-Aldrich</td><td>F4135-500</td><td></td></tr><tr><td>LIF</td><td>EMD Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140148</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Methyl Viologen dichloride (Paraquat)</td><td>Sigma-Aldrich</td><td>856177</td><td></td></tr><tr><td>Dimethyl Sulphoxide Hybri-MAX</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>EmbryoMAX 0.1% gelatin</td><td>EMD Millipore</td><td>ES006B</td><td></td></tr><tr><td>DPBS/Modified</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>T25 Flask</td><td>Corning</td><td>353108</td><td></td></tr><tr><td>T75 flask</td><td>Corning</td><td>353135</td><td></td></tr><tr><td>100-mm&amp;nbsp; plate</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>150-mm&amp;nbsp; plate</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>96-well&amp;nbsp; plate</td><td>Corning</td><td>3585</td><td></td></tr><tr><td>96-well U-bottom plate</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>50-ml reservoir&amp;nbsp;</td><td>Corning</td><td>4870</td><td></td></tr><tr><td>15-ml tubes</td><td>VWR International, LLC</td><td>82050-276</td><td></td></tr><tr><td>Primary Mouse Embryonic Fibroblasts</td><td>EMD Millipore</td><td>PMEF-NL</td><td></td></tr><tr><td>DR4 Mouse Embryonic Fibroblasts</td><td>Applied StemCell</td><td>ASF-1001</td><td></td></tr><tr><td>Mitomycin C</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP25312</td><td></td></tr><tr><td>Geneticin (G418)</td><td>Life Technologies</td><td>11811-023</td><td></td></tr><tr><td>Doxycycline</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP26531</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td>377267</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5702</td><td></td></tr><tr><td>TC10 cell counter</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counting Slides (for TC10)</td><td>Bio-Rad</td><td>1450011</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electroporation&lt;/strong&gt;</td></tr><tr><td>Gene Pulser Xcell</td><td>Bio-Rad</td><td>1652611</td><td></td></tr><tr><td>Gene Pulser Cuvettes (4&amp;#160;mm gap)</td><td>Bio-Rad</td><td>1652088</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Molecular Biology&lt;/strong&gt;</td></tr><tr><td>Thermal Cycler</td><td>Eppendorf</td><td>Mastercylcer ep Gradient&amp;nbsp; S</td><td></td></tr><tr><td>Puregene Core kit B</td><td>Qiagen</td><td>158745</td><td></td></tr><tr><td>Topo-TA Cloning kit</td><td>Life Technologies&amp;nbsp;</td><td>450030</td><td></td></tr><tr><td>High Capacity cDNA synthesis kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>NaCl</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP358-212</td><td></td></tr><tr><td>100% ethanol</td><td>Decon Laboratories, Inc.</td><td>2716</td><td></td></tr><tr><td>Double Processed Tissue Culture Water</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>Sau3A1</td><td>New England BioLabs</td><td>R0169L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202T</td><td></td></tr><tr><td>EcoRV</td><td>New England BioLabs</td><td>R3195S</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;96-well Lysis Buffer (Ramires-Solis &lt;em&gt;et al.&lt;/em&gt; 1992)&lt;/strong&gt;</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher BioReagents&amp;nbsp;</td><td>A144-212</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L5777</td><td></td></tr><tr><td>Proteinase-K</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1700</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Electrophoresis&lt;/strong&gt;</td></tr><tr><td>Mini-Sub Cell GT</td><td>Bio-Rad</td><td>170-4469EDU</td><td></td></tr><tr><td>LE Agarose&amp;nbsp;</td><td>GeneMate</td><td>E3120500</td><td></td></tr><tr><td>Ethidium Bromide&amp;nbsp;</td><td>Fisher BioReagents&amp;nbsp;</td><td>BP1302</td><td></td></tr><tr><td>100 BP DNA Ladder</td><td>New England BioLabs</td><td>N3231S</td><td></td></tr><tr><td>1 Kb DNA Ladder</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr><tr><td>2-log DNA Ladder</td><td>New England BioLabs</td><td>N3200L</td><td></td></tr></tbody></table>

forward genetic approach to uncover stress resistance genes in mice &#8212; a high-throughput screen in es cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

In a typical mutagenesis experiment, the transfection consists of a total of 3 x 107 ES cells with PB-UPA and mPB transposase. The numbers of gene-trapped ES cells generated in two independent experiments are summarized in Table 1. The efficiency of gene-entrapment is about 0.04%. The combined gene-trap libraries contain 22,400 independent mutants, about a full coverage of the mouse genome (23,000 coding genes). Excessive coverage could be achieved by generating more mutants through repeated mutagenesis and/or scaling up the process.
Because the gene-trap libraries contain random mutations, it is not possible to predict the number of mutants possessing a stress resistance phenotype. Two independent selections for PQ resistance were performed; the combined screening of 22,440 independent gene-trap mutations uncovered 17 mutations that confer cellular resistance to PQ (0.08%) (Table 1).
We have purified 3 mutant clones, the Pigl, Tiam1, and Rffl, from the master plates for further analysis and mouse production. Cells with heterozygous Rffl mutation were successfully induced to produce homozygous progeny by knocking out Blm. However, we failed to produce any homozygous mutants from the Pigl and Tiam1 mutants, presumably due to a homozygosity-linked cell lethality. The stress resistance of these purified clones was confirmed by using 2 different types of stressors: omission of 2-mercaptoethanol (2-ME) and PQ (Figure&#160;2). Upon removing the PB from these clones, the associated stress resistance phenotype was also lost (Figure&#160;2), confirming that the gene-trap mutations are the causal factor. Characterization of clones recovered from the master plates is crucial as it would rule out stochastic lesion during stress selection as the cause of the observed phenotype.
Out of 3 mutant ES cells being introduced into blastocysts for mouse production (Table 2), 2 lines (Pigl and Tiam1) show germline transmission. The Rffl injection produced only one female chimera which was not an optimal number of chimera to begin with. Thus, the failure of germline transmission of Rffl is likely due to one incompetent chimera. We tested the cellular phenotype of skin fibroblasts isolated from the Pigl mutant mice and showed that they maintained the reduced level of endogenous reactive oxygen species (ROS) as well as stress resistance to PQ (Figure&#160;3).
<img alt="Figure 1" src="/files/ftp_upload/53062/53062fig1.jpg" /> 
Figure 1. ES cell mutagenesis and stress selection. (A) PB-UPA vector. An unbiased polyA (UPA) trap vector comprising of a splice acceptor (SA), bovine growth hormone poly-adenylate signal (pA), phosphoglycerate kinase (PGK) promoter, neomycin phosphotransferase (neo), internal ribosomal entry site (IRES), and a splice donor (SD) with artificial ATG (in 3 different reading frames) was cloned into the piggyBac transposon, flanked by the 3&#8217; and 5&#8217; long terminal repeat (LTR). (B) Random mutagenesis of ES cells and selection for stress resistant clones. ES cells were co-transfected by electroporation with PB-UPA and mPBase followed by plating onto 96-well plates. Gene trapped clones were selected by G418 resistance; once confluent, they were divided into 2 replica sets. One replica set was further divided into two half for DNA isolation and stress selection by paraquat (PQ); the other replica set (master) was frozen down. The surviving ES cell colonies recovered from the stress treatment were analyzed molecularly for the PB insertion by Sp-PCR. Primers were then designed to screen the replica DNA plate from which the well containing the sibling PQR clone could be located on the master plate. These cells are for stress resistance assay on different stressors and for mouse production. Modified from Chick et al. 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53062/53062fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53062/53062fig2.jpg" /> 
Figure 2. Stress resistance in mutant ES cells. (A) Resistance to 2-mercaptoethanol (2-ME) withdrawal. (B) Resistance to paraquat (PQ). Shown are the parental wild-type ES cells (C9: yellow), a stress-resistant control ES cell clone, (4C11: red), recovered from a previous study 5, and three gene-trap clones (grey). Cells were subjected to stress treatment for two days, after which the number of viable cells was counted. Pigl, Tiam1, and Rffl heterozygotes (PB/+: dark grey) exhibit resistance to both of these stressors. Rffl homozygotes (PB/PB: black) exhibit stronger stress resistance compared to the heterozygotes. Resistance to both stressors was lost when the PB insertions were removed (+/+: light grey). Error bars represent SD of mean (n=4). * P &#60; 0.001, # P = 0.01 between the gene-trap clones and the wild-type parental C9, evaluated by Student&#8217;s t-test. (C) ES cell colony formation under PQ (10 &#181;M) treatment. Stress-resistant ES clones were able to form colonies in culture under PQ treatment while the number and size of colonies from the wild-type clone were significantly reduced. Modified from Chick et al. 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53062/53062fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53062/53062fig3.jpg" /> 
Figure 3. Characterization of Pigl PB/+ fibroblasts. (A) Reactive oxygen species (ROS) level. Tail skin fibroblasts isolated from wild-type (WT) and Pigl PB/+ mice were stained with CM-DCFCA and the fluorescence was normalized against Hoechst. The ROS content was expressed as relative fluorescence unit (RFU). The P value was evaluated by two tailed Student&#8217;s t-test (n=8). (B) PQ resistance. Tail skin fibroblasts from wild-type (WT) and PiglPB/+ mice were exposed to 4 mM PQ for 6 hr, after which the viability of the cells were measured by MTT assays. The percentage of live cells after PQ treatment was calculated by the ratio of the absorbance obtained from the PQ-treated cells and that from the un-treated cells. The P value was evaluated by one tailed Student&#8217;s t-test (n=8). Modified from Chick et al. 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53062/53062fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
&#160;&#173;&#160;
<table border="0" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Library</td>
			<td>Sub-libraries</td>
			<td>ES cell strain</td>
			<td>No. of gene trapped clone</td>
			<td>No. of PQR clones</td>
		</tr>
		<tr>
			<td>1</td>
			<td>C9PA01 &#8211; C9PA10</td>
			<td>C9 (Blm tet/tet)</td>
			<td>9,000</td>
			<td>7</td>
		</tr>
		<tr>
			<td>2</td>
			<td>C9PA11 &#8211; C9PA20</td>
			<td>C9 (Blm tet/tet)</td>
			<td>13,440</td>
			<td>10</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Total</td>
			<td>22,440</td>
			<td>17</td>
		</tr>
	</tbody>
</table>
Table 1. PiggyBac gene-trap library construction and the recovery of PQ resistant clones. Modified from Chick et al. 7.
&#160;&#173;&#160;
<table border="0" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>ES cell clone injected</td>
			<td>No. of chimera recovered</td>
			<td>Germline transmission *</td>
		</tr>
		<tr>
			<td>Pigl PB/ Pigl +</td>
			<td>4</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Tiam1 PB / Tiam1 +</td>
			<td>2</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Rffl PB / Rffl +</td>
			<td>1</td>
			<td>No</td>
		</tr>
	</tbody>
</table>
Table 2. Generation of mice. Modified from Chick et al. 7.
* Germline transmission was confirmed by inheritance of the gene-trap allele detected by PCR genotyping.

Watch this Scientific Journal Video about Forward Genetic Approach to Uncover Stress Resistance Genes in Mice Ã¢â‚¬â€ A High-throughput Screen in ES Cells at JoVE.com

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K Views.  University of Colorado Denver. Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Video: Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Genetics

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Biology

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...

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.

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.

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

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

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Chapters in this video

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

High-throughput Yeast Plasmid Overexpression Screen

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

Measurements of Physiological Stress Responses in C. Elegans

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe