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).

1. Gene-trapped ES Cell Library Construction Using piggyBac Transposon
<ol>
	<li>Prepare primary mouse embryonic fibroblasts (PMEF) as feeders for ES cell culture
	<ol>
		<li>Thaw one vial of mitomycin C-inactivated PMEF (5.0 x 106) in a 37 &#176;C water bath. Transfer the cells into 5 ml of ES cell medium (DMEM containing 15% FBS, 1,000 unit/ml of leukemia inhibitory factors, 100 &#181;M nonessential amino acids, 2 mM glutamine, 55 &#181;M 2-mercaptoethanol, and 25 unit/ml penicillin/streptomycin),...

<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>

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 a...

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 border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vector</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PB-UPA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPBase</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPBasePuro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500-ml Stericup filters</td>
			<td>EMD Millipore</td>
			<td>SCGPU05RE</td>
		</tr>
		<tr>
			<td>250-ml Stericup filters</td>
			<td>EMD Millipore</td>
			<td>SCGPU02RE</td>
		</tr>
		<tr>
			<td>50-ml Steriflip-GV filters</td>
			<td>EMD Millipore</td>
			<td>SE1M179M6</td>
		</tr>
		<tr>
			<td>KO DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Tissue Culture Biologicals</td>
			<td>104</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>EMD Millipore</td>
			<td>ESG1107</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140148</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
		</tr>
		<tr>
			<td>Methyl Viologen dichloride (Paraquat)</td>
			<td>Sigma-Aldrich</td>
			<td>856177</td>
		</tr>
		<tr>
			<td>Dimethyl Sulphoxide Hybri-MAX</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
		</tr>
		<tr>
			<td>EmbryoMAX 0.1% gelatin</td>
			<td>EMD Millipore</td>
			<td>ES006B</td>
		</tr>
		<tr>
			<td>DPBS/Modified</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
		</tr>
		<tr>
			<td>T25 Flask</td>
			<td>Corning</td>
			<td>353108</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Corning</td>
			<td>353135</td>
		</tr>
		<tr>
			<td>100-mm&#160; plate</td>
			<td>Corning</td>
			<td>353003</td>
		</tr>
		<tr>
			<td>150-mm&#160; plate</td>
			<td>Corning</td>
			<td>430599</td>
		</tr>
		<tr>
			<td>96-well&#160; plate</td>
			<td>Corning</td>
			<td>3585</td>
		</tr>
		<tr>
			<td>96-well U-bottom plate</td>
			<td>Corning</td>
			<td>3799</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3526</td>
		</tr>
		<tr>
			<td>50-ml reservoir&#160;</td>
			<td>Corning</td>
			<td>4870</td>
		</tr>
		<tr>
			<td>15-ml tubes</td>
			<td>VWR International, LLC</td>
			<td>82050-276</td>
		</tr>
		<tr>
			<td>Primary Mouse Embryonic Fibroblasts</td>
			<td>EMD Millipore</td>
			<td>PMEF-NL</td>
		</tr>
		<tr>
			<td>DR4 Mouse Embryonic Fibroblasts</td>
			<td>Applied StemCell</td>
			<td>ASF-1001</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP25312</td>
		</tr>
		<tr>
			<td>Geneticin (G418)</td>
			<td>Life Technologies</td>
			<td>11811-023</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP26531</td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>377267</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5702</td>
		</tr>
		<tr>
			<td>TC10 cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Slides (for TC10)</td>
			<td>Bio-Rad</td>
			<td>1450011</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell</td>
			<td>Bio-Rad</td>
			<td>1652611</td>
		</tr>
		<tr>
			<td>Gene Pulser Cuvettes (4 mm gap)</td>
			<td>Bio-Rad</td>
			<td>1652088</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>Mastercylcer ep Gradient&#160; S</td>
		</tr>
		<tr>
			<td>Puregene Core kit B</td>
			<td>Qiagen</td>
			<td>158745</td>
		</tr>
		<tr>
			<td>Topo-TA Cloning kit</td>
			<td>Life Technologies&#160;</td>
			<td>450030</td>
		</tr>
		<tr>
			<td>High Capacity cDNA synthesis kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP358-212</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2716</td>
		</tr>
		<tr>
			<td>Double Processed Tissue Culture Water</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
		</tr>
		<tr>
			<td>Sau3A1</td>
			<td>New England BioLabs</td>
			<td>R0169L</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202T</td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England BioLabs</td>
			<td>R3195S</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Lysis Buffer (Ramires-Solis et al. 1992)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher BioReagents&#160;</td>
			<td>A144-212</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>L5777</td>
		</tr>
		<tr>
			<td>Proteinase-K</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP1700</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT</td>
			<td>Bio-Rad</td>
			<td>170-4469EDU</td>
		</tr>
		<tr>
			<td>LE Agarose&#160;</td>
			<td>GeneMate</td>
			<td>E3120500</td>
		</tr>
		<tr>
			<td>Ethidium Bromide&#160;</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP1302</td>
		</tr>
		<tr>
			<td>100 BP DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3231S</td>
		</tr>
		<tr>
			<td>1Kb DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3232S</td>
		</tr>
		<tr>
			<td>2-log DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3200L</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 m...

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 ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â 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.

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 ...

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Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...