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 a...

Langlebigkeit hat eine intime Beziehung mit Stressresistenz. Im allgemeinen langlebige Spezies weisen häufig erhöhte Resistenz gegen multiple Stressoren, wie Wasserstoffperoxid, Paraquat (PQ), UV, Wärme und Schwermetalle 1,2. Im Gegensatz dazu erhöhte Empfindlichkeit gegenüber Stress verursacht verkürzten Lebensdauer und / oder eine Krankheit anfälligen Phänotyp zusagen. Die Antioxidationsmittel fang Weg war lange spekuliert worden, eine wichtige Rolle bei der Verleihung Stressresistenz für das Tier zu spielen. Mit wenigen Ausnahmen, Studien aus einer Vielzahl von transgenen Tieren mit Manipulationen in verschiedenen antioxidativen Enzymen (zB SOD) zeigen jedoch, dass die Erhöhung des Oxidationsfänger Enzyme nicht die Lebensdauer oder die Gesundheit Spannweite 3 zu erhöhen. Diese Daten legen nahe, dass die Stressresistenz Merkmal konsequent in langlebigen Tieren beobachtet wird von anderen zellulären Wege noch aufgedeckt werden vermittelt. Wir nahmen eine unvoreingenommene vornegenetischer Ansatz für Gene, die auf mutierten, eine Stressresistenz-Phänotyp in kultivierten embryonalen Stammzellen (ES-Zellen) verleihen könnten. ES-Zellen bieten zwei wesentliche Vorteile in dieser Studie: (1) anspruchsvolle genetische Manipulationen sind vorhanden, um das Genom von ES-Zellen zu modifizieren; und (2) Stress resistente ES-Zellen aus dem Bildschirm wiedergewonnen können direkt für Maus Produktion verwendet werden, was eine schnelle Übersetzung in ganzen Tierstudien zur Lebenserwartung und Gesundheitsspanne zu messen. In diesem Bericht beschrieben wir die Verwendung von C9 ES-Zelllinie, in welcher die BLM Allele unter Steuerung durch ein Tetracyclin ansprechende Element. Behandlung von Doxycyclin (DOX) vorübergehend von der Expression von Blm führt zu einer erhöhten Vorfall von Schwesterchromatidaustausch gedreht. Diese kurzfristige Blm Knock-out für die Erzeugung homozygoter Mutationen innerhalb der heterozygote Population erlaubt, so dass rezessive Mutationen für Stressresistenz könnte in das Screening-Verfahren erfasst werden. Wirbeschrieben die Verwendung von piggyBac (PB) Transposon als erbgutverändernd nach dem Zufallsprinzip setzen Sie einen Poly-A-Trap-Kassette (PB-UPA), um Gene in das Genom zu mutieren. Zellen, die mit Unterbrechung eines Gens durch die Poly-A-Falle wurden G418-resistente und konnte zurückgewonnen, so dass eine Sammlung von Gen-Trap-Mutanten (Gen-Trap-Bibliothek) können hergestellt werden, und in der Folge für die mutierten Klone, die Stress resistent waren zu sehen sein. Stress-resistente Klone von der Selektion gewonnen könnte ziemlich rasch durch molekulare Techniken hinsichtlich der Anzahl von Einfügungen (qPCR) charakterisiert werden, der Ort der Insertion (splinkerette PCR), die Identität der unterbrochene Gen (BLAST) und sein Expressionsniveau ( RT-qPCR). Die PB Einsetzen könnte durch transiente Expression mPB Transposase in dem Klon wieder mobilisiert werden, um die Wildtyp-DNA-Sequenz wieder herzustellen und damit zu testen für den Verlust von Stressresistenz. Diese sind leistungsfähige Möglichkeiten, um die Kausalität der Mutation zu bestätigen, die vor getan werden sollteteure Maus Produktion. Frühere Studien haben gezeigt, dass Zellen, die Stressfaktoren ausgesetzt verloren ihre Pluripotenz 4,5. Somit wird in diesem Protokoll ist die Erhaltung einer Replikatgruppe von mutierten Zellen, die nicht mit Stressoren behandelt werden, würde die für den erfolgreichen Produktions Maus. Unser Labor hat den C9 ES-Zelllinie und der PB-UPA-Vektor entwickelt, die beide zur Verfügung stehen, um anderen Forschern auf Anfrage. Das Protokoll die hier berichtet wird durch die Erzeugung von de novo Bibliothek von Gen-gefangen ES-Zellen mit PB-UPA (1A), gefolgt von Replika-Plattierung und Streßselektion auf Stress-resistente Klone (1B) zu isolieren, zu starten. Wir haben gezeigt, die Auswahl mit Paraquat, ein potenter Radikalerzeuger innerhalb der Zellen. Praktisch jede zytotoxische Verbindung oder ein Toxin, beispielsweise ER Stressoren (zB Thapsigargin und Tunicamycin), neuronale Oxidationsmittel (zB MPP +, 6-Hydroxy Dopamin und Rotenon), Wärme und SchwerMetalle (zB Cd, Se) könnten mit dem Verfahren geeignet ist, für die jeweiligen resistenten Mutanten auszuwählen.

<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 einem typischen Experiment Mutagenese erfolgte die Transfektion besteht aus einer Gesamtzahl von 3 x 10 7 ES-Zellen mit PB-UPA und mPB Transposase. Die Zahl der Gene-gefangen ES-Zellen in zwei unabhängigen Experimenten erzeugt, sind in Tabelle 1 zusammengefasst. Die Effizienz der Gen-Einschluss etwa 0,04%. Die kombinierten Gen-Trap-Bibliotheken enthalten 22.400 unabhängigen Mutanten, über eine vollständige Abdeckung des Mausgenoms (23.000 kodierenden Gene). Übermäßi...

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

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

Stressresistenz ist eines der Markenzeichen für Langlebigkeit und ist bekannt, genetisch geregelt. Hier entwickelten wir eine unvoreingenommene Hochdurchsatzverfahren für Mutationen, die Stress-Resistenz verleihen in ES-Zellen, mit dem Maus-Modelle für Langlebigkeit Studien entwickeln zu screenen.

Vorwärts genetische Ansatz zur Stressresistenz-Gene in Mäuse Entdecken - Hochdurchsatz-Bildschirm in ES-Zellen

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

forward-genetic-approach-to-uncover-stress-resistance-genes-mice-high

Research

JoVE Journal

Developmental Biology

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

8.1K Aufrufe.  University of Colorado Denver.  Stressresistenz ist eines der Markenzeichen für Langlebigkeit und ist bekannt, genetisch geregelt. Hier entwickelten wir eine unvoreingenommene Hochdurchsatzverfahren für Mutationen, die Stress-Resistenz verleihen in ES-Zellen, mit dem Maus-Modelle für Langlebigkeit Studien entwickeln zu screenen. 

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

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Ein optogenetische Ansatz zur Abschätzung Bildung neuronaler Verbindungen in einer Co-Kultursystem

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forschung

Entwicklungsbiologie

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technik auf Zielmikroinjektions an die Entwicklungs<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Eine einfache Methode zur Identifizierung von Kinasen, die die embryonale Stammzell-Pluripotenz durch Hochdurchsatz-Inhibitor-Screening regulieren

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Eine neuartige Mamma-Fat-Pad-Transplantationstechnik zur Visualisierung der Gefäßerzeugung von vaskulären endothelialen Stammzellen

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Ein standardisierter Ansatz für Multispezies Reinigung von männlichen männlichen Keimzellen durch mechanische Gewebedissoziation und Durchflusszytometrie

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Anwendung von RNAi und Hitze-Schock-induzierte Transkription Faktor Ausdruck zu programmieren Keimzellen Neuronen in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualisierung der Knoten und Notochordal Platte In Gastrulating Maus-Embryonen mittels Rasterelektronenmikroskopie und ganze Mount Immunfluoreszenz

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In vitro-Generation der Somiten Derivate aus menschlichen induzierten Zellen pluripotenten Stammzellen

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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 health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

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

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Bewertung von oxidativen Schäden in den primären Maus-Augen-Oberflächenzellen/Stammzellen als Reaktion auf Uv-C-Schäden (UV-C)

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

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Durchführung von Human Skeletal Muscle Xenografts bei immundefizienten Mäusen

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...