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.

יש אריכות ימים מערכת יחסים אינטימיות עם התנגדות ללחץ. באופן כללי, מיני חיים ארוכים לעתים קרובות להפגין גדלו התנגדות ללחצים מרובים, כגון מי חמצן, paraquat (PQ), UV, חום, ומתכות כבדות 1,2. לעומת זאת, רגישות מוגברת ללחץ נוטה לנבא תוחלת חיים מקוצרים ו / או פנוטיפ מחלה נוטה יותר. מסלול הדחה נוגד החמצון כבר זמן רב העריך לשחק תפקיד מרכזי בהענקת התנגדות מתח לבעלי החיים. עם זאת, עם כמה יוצאים מן הכלל, מחקרים ממגוון רחב של בעלי חיים מהונדסים עם מניפולציות באנזימים שונים נוגד חמצון (לדוגמא, SOD) מצביעים על כך שהגדלת רמת אנזימי הדחה חמצון אינו מגבירה את תוחלת חיים או בריאות תוחלת 3. נתונים אלה מצביעים על כך שתכונת התנגדות המתח נצפתה באופן עקבי בבעלי חיים חיים ארוכים מתווכת על ידי מסלולים סלולריים אחרים עדיין לא נחשפו. לקחנו קדימה משוחדגישה גנטית לזיהוי גנים, אשר על מוטציה, יכול להעניק הפנוטיפ התנגדות מתח בתאי גזע עוברי בתרבית (ES). תאי גזע עובריים מציעים שני יתרונות עיקריים במחקר זה: (1) מניפולציות גנטיות מתוחכמות זמינות כדי לשנות את הגנום של תאי גזע עובריים; ו- (2) בכל תאי גזע עובריים עמידים לחץ התאוששו מהמסך ניתן להשתמש ישירות לייצור עכבר, המאפשרים תרגום מהיר למחקרים בבעלי חיים שלמים כדי למדוד את תוחלת חיים ותוחלת בריאות. בדו&quot;ח זה, שתארנו את השימוש בקו תא C9 ES, שבו אללים BLM היו בשליטה על ידי אלמנט תגובה טטרציקלין. הטיפול בדוקסיציקלין (DOX) כבוי זמני הביטוי של BLM המוביל לאירוע מוגבר של בורסה כרומטידה אחות. לטווח קצר זה BLM נוק-אאוט אפשר לדור של מוטציות הומוזיגוטים בתוך אוכלוסיית heterozygote כך שמוטציות רצסיבי להתנגדות ללחץ יכולות להיות שנתפסו בתהליך המיון. אנחנוגם תאר את השימוש בpiggyBac transposon (PB) כmutagen להכניס באופן אקראי פולי-קלטת מלכודת (PB-UPA) להשתנות גנים בגנום. תאים עם הפרעה של גן על ידי פולי-מלכודת הפכו G418 עמידים ויכולים להיות התאוששו, כך שאוסף של מוטציות גנטי מלכודת (ספריית גן-מלכודת) יכול להתבצע, ולאחר מכן הוקרן לשיבוטי מוטציה שהיו מתח עמיד. שיבוטים עמידים מתח התאוששו מהבחירה יכולים להיות מאופיינים ולא במהירות על ידי טכניקות מולקולריות בהקשר של מספר ההוספות (qPCR), האתר של הכנסה (splinkerette PCR), את זהותו של הגן שיבש (פיצוץ), ורמת הביטוי שלה ( RT-qPCR). הכנסת PB יכולה להיות remobilized על ידי ביטוי חולף של transposase MPB בשיבוט כדי לשחזר את רצף ה- DNA wild-type ובכך לבדוק את אובדן התנגדות לחץ. אלה הם דרכים רבות עוצמה כדי לאשר סיבתיות של המוטציה, אשר צריך להיעשות לפנילייצור עכבר יקר. מחקרים קודמים הראו כי תאים שנחשפו ללחצים איבדו 4.5 pluripotency. כך, בפרוטוקול זה, השמירה על העתק קבוצה של תאים שעברו מוטציה, שלא להיות מטופלים עם גורמי לחץ היא קריטית לייצור עכבר מוצלח. המעבדה שלנו מהונדסת קו תא C9 ES ווקטור PB-UPA, שניהם זמינות לחוקרים אחרים על פי דרישה. הפרוטוקול דיווח כאן יתחיל בדור של ספריית דה נובו של תאים לכודים גן ES עם PB-UPA (איור 1 א), ואחריו ציפוי העתק ובחירת מתח כדי לבודד שיבוטים לחץ עמידים (איור 1). אנחנו הוכחנו את הבחירה בparaquat, גנרטור רדיקלים חופשי חזק בתוך תאים. כמעט, כל מתחם ציטוטוקסיות או רעלן, למשל, גורמי לחץ ER (למשל, thapsigargin וtunicamycin), חמצון עצבי (למשל, MPP +, דופמין 6-הידרוקסי, וrotenone), חום, וכבדמתכות (למשל, CD, Se), יכולות להיות מותאמות לשיטה כדי לבחור למוטציות עמידות בהתאמה.

<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.

בניסוי mutagenesis טיפוסי, transfection מורכב מסכום כולל של 3 x 10 7 תאי גזע עובריים עם PB-UPA וtransposase MPB. המספרים של תאי גזע עובריים-לכוד גן שנוצרו בשני ניסויים בלתי תלויים מסוכמים בטבלה 1. היעילות של גן-מלכוד היא% על 0.04. ספריות גן-מלכודת המשולבת מכילות 22,400 מוטציות עצמאיות, על כיסוי מלא של גנום העכבר (23,000 גני קידוד). כיסוי מוגזם יכול להיות מושגת על ידי יצירה יותר מוטציות באמצעות mutagenesis חזר ו / או דרוג את התהליך. בגלל ספריות גן-מלכודת מכילות מוטציות אקראיות, לא ניתן לחזות את מספר מוטציות בעלי פנוטיפ התנגדות לחץ. שתי בחירות עצמאיות להתנגדות PQ בוצעו; ההקרנה בשילוב של 22440 מוטציות גנטי מלכודת עצמאיות נחשפה 17 מוטציות שמקנות עמידות לסלולרית PQ (0.08%) (טבלת 1). &lt;p class = &quot;jove_content&quot;&gt; יש לנו מטוהר 3 שיבוטים מוטציה, Pigl, Tiam1, וRffl, מהצלחות האב לניתוח נוסף וייצור עכבר. תאים עם המוטציה Rffl הטרוזיגוטיים היו מושרה בהצלחה לייצר צאצאים הומוזיגוטים על ידי לדפוק את BLM. עם זאת, אנחנו לא הצליחו לייצר שום מוטציות הומוזיגוטים ממוטציות Pigl וTiam1, ככל הנראה בשל קטלני תא צמוד homozygosity. ההתנגדות ללחץ של שיבוטים המטוהרים אלה אושרה על ידי שימוש 2 סוגים שונים של לחץ: השמטה של 2-mercaptoethanol (2-ME) וPQ (איור 2). עם הסרת PB משיבוטים אלה, הפנוטיפ התנגדות הלחץ קשור גם איבד (איור 2), המאשר כי המוטציות גנטי המלכודת הן הגורם סיבתי. אפיון של שיבוטים התאוששו מהצלחות ההורים הוא חיוני כפי שהיה פוסל את נגע סטוכסטיים במהלך בחירת מתח כגורם לפנוטיפ הנצפה. מתוך 3תאי גזע עובריים מוטציה שהוצגו לblastocysts לייצור עכבר (טבלה 2), 2 קווים (Pigl וTiam1) להראות שידור germline. הזרקת Rffl מיוצרת הכימרה נקבה יחידה שלא היה מספר אופטימלי של הכימרה מלכתחילה. לפיכך, הכישלון של העברת germline של Rffl עשוי הכימרה בשל אחד פסול. בדקנו את הפנוטיפ הסלולרי של fibroblasts העור מבודד מהעכברים שעברו המוטציה Pigl והראיתי שהם שמרו על הרמה המופחתת של מיני חמצן מגיבים אנדוגני (ROS), כמו גם התנגדות ללחץ PQ (איור 3). <img alt="איור 1" src="/files/ftp_upload/53062/53062fig1.jpg" /> איור 1. ES mutagenesis תא ובחירת מתח. וקטור () PB-UPA. וקטור הפולה מלכודת (UPA) משוחד הכולל acceptor אחוי (SA), אות פולי-adenylate הורמון גדילת שור (הרשות הפלסטינית), phosphogקינאז lycerate אמרגן (PGK), phosphotransferase נאומיצין (ניאו), אתר כניסת ריבוזומלי הפנימי (IRES), ותורם אחוי (SD) עם ATG המלאכותי (ב 3 מסגרות קריאה שונות) שובט לתוך transposon piggyBac, מוקף 3 &#39; וחזור על 5 &#39;מסוף ארוך (LTR). (ב) mutagenesis אקראי של תאי גזע עובריים ובחירה לשיבוטים עמידים לחץ. תאי גזע עובריים היו שיתוף transfected ידי electroporation עם PB-UPA וmPBase אחרי ציפוי על 96-גם צלחות. שיבוטים לכודים גן נבחרו על ידי התנגדות G418; פעם אחת ומחוברות, הם חולקו לקבוצות 2 העתק. סט העתק אחד לשתי מחצית לבידוד DNA ובחירת מתח על ידי paraquat (PQ); הסט האחר ההעתק (אב) היה קפוא במורד. מושבות תאי הגזע העובריים ששרדו התאוששו מטיפול הלחץ נותחו מולקולריות להחדרת PB על ידי Sp-PCR. Primers אז נועד להקרין את צלחת ה- DNA ההעתק ממנו מכילה גם sibliשיבוט ng PQ R יכול להיות ממוקם על צלחת האדון. תאים אלה הם עבור assay התנגדות לחץ על גורמי לחץ שונים ולייצור עכבר. שונה מהאפרוח et al. 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53062/53062fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/53062/53062fig2.jpg" /> התנגדות 2. מתח איור בתאי גזע עובריים מוטציה. התנגדות () 2-mercaptoethanol נסיגה (2-ME). (ב) התנגדות לparaquat (PQ). מוצגים הם תאי ההורים wild-type ES (C9: צהוב), שיבוט תאי גזע עובריים שליטה עמידה לעקות, (4C11: אדום), התאוששו ממחקר קודם 5, ושלושה שיבוטים גנטי מלכודת (אפורים). תאים היו נתונים ללחץ טיפול במשך יומיים, לאחר שמספר תאי קיימא נספר. &lt;<html> em&gt; heterozygotes Pigl, Tiam1, וRffl. (PB / +: אפור כהה) התנגדות תערוכה לשניהם הלחץ הללו הומוזיגוטים Rffl (PB / PB: שחור) להפגין התנגדות ללחץ חזקה בהשוואה לheterozygotes. התנגדות לשני גורמי הלחץ אבדה כאשר הוספות PB הוסרו (+ / +: אפורה בהירה). ברים שגיאה מייצגים SD של ממוצע (n = 4). * P &lt;0.001, p = 0.01 # בין השיבוטים גנטי המלכודת וC9 ההורים wild-type, הוערכו על ידי -test t של הסטודנט. הקמת מושבה (C) ES תא תחת PQ טיפול (10 מיקרומטר). השיבוטים ES עמידים לעקות היו מסוגלים ליצור מושבות בתרבות תחת טיפול PQ תוך המספר והגודל של מושבות משיבוט wild-type הופחתו באופן משמעותי. שונה מהאפרוח et al. 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53062/53062fig2large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> NT &quot;FO: לשמור-together.within עמודים =&quot; תמיד &quot;&gt; <img alt="איור 3" src="/files/ftp_upload/53062/53062fig3.jpg" /> איור 3. אפיון של Pigl PB / + fibroblasts. רמה () מיני חמצן תגובתי (ROS). fibroblasts עור זנב מבודד מwild-type (WT) וPigl PB / + העכברים הוכתמו CM-DCFCA והקרינה הייתה מנורמל נגד Hoechst. תוכן ROS בא לידי ביטוי כיחידה יחסי הקרינה (RFU). ערך P הוערך על ידי -test t שני סטודנטים זנב (n = 8). התנגדות (ב) PQ. fibroblasts עור זנב מwild-type (WT) וPigl PB / + העכברים נחשפו ל4 מ&quot;מ PQ עבור שעה 6, לאחר שהכדאיות של התאים נמדדו על ידי מבחני MTT. אחוז של תאי חיים לאחר טיפול PQ היה מחושב על ידי היחס של הספיגה המתקבל מהספירה שטופלה PQLLS וכי מהתאים שטופל האו&quot;ם. ערך P הוערך על ידי -test t של סטודנט אחד הזנב (n = 8). שונה מהאפרוח et al. 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53062/53062fig3large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <table border="0" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"><tbody><tr><td> סִפְרִיָה </td><td> תת-ספריות </td><td> מתח בתאי גזע עובריים </td><td> מס &#39;של שיבוט גן לכוד </td><td> מס &#39;של שיבוטים PQ R </td></tr><tr><td> 1 </td><td> C9PA01 - C9PA10 </td><td> C9 (BLM ט / ט) </td><td> 9,000 </td><td> 7 </td></tr><tr><td> 2 </td><td> C9PA11 - C9PA20 </td><td> C9 (BLM ט / ט) </td><td> 13440 </tד&gt; <td> 10 </td></tr><tr><td></td><td></td><td> סה&quot;כ </td><td> 22440 </td><td> 17 </td></tr></tbody></table> שולחן בניית 1. PiggyBac גן-מלכודת ספרייה וההתאוששות של שיבוטים עמידים PQ. השתנה מet al צ&#39;יק. 7. <table border="0" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שיבוט תאי גזע עובריים שהוחדר </td><td> מס &#39;של הכימרה התאושש </td><td> שידור germline * </td></tr><tr><td> Pigl PB / Pigl + </td><td> 4 </td><td> כן </td></tr><tr><td> Tiam1 PB / Tiam1 + </td><td> 2 </td><td> כן </td> &lt;/ Tr&gt; <tr><td> Rffl PB / Rffl + </td><td> 1 </td><td> לֹא </td></tr></tbody></table> דור טבלה 2. של עכברים. השתנה מet al צ&#39;יק. 7. * שידור germline אושר על ידי ירושה של אלל גן-המלכודת זוהה על ידי genotyping PCR.

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

התנגדות מתח היא אחד מסימני ההיכר לאריכות ימים, והוא ידוע להיות נשלטת גנטי. כאן, פיתחנו שיטת תפוקה גבוהה משוחדת למסך למוטציות המעניקות התנגדות מתח בתאי גזע עובריים שבה לפתח מודלים של עכברים ללימודי אריכות ימים.

גישה גנטית קדימה ללחשוף גני התנגדות מתח בעכברים - מסך תפוקה גבוהה בתאי גזע עובריים

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.  התנגדות מתח היא אחד מסימני ההיכר לאריכות ימים, והוא ידוע להיות נשלטת גנטי. כאן, פיתחנו שיטת תפוקה גבוהה משוחדת למסך למוטציות המעניקות התנגדות מתח בתאי גזע עובריים שבה לפתח מודלים של עכברים ללימודי אריכות ימים. 

Video: Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — 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.

גישת optogenetic להערכת יצירת קשרים עצביים במערכת Co-תרבות

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

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.

טכניקה לכוון Microinjection אל פיתוח<em&gt; Xenopus</em&gt; כליה

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 ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

הערכה סימולטני של DNA Cardiomyocyte סינתזה ploidy: שיטה Assist כימות Cardiomyocyte רגנרציה מחזור

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

ביים הבידול של אבות Hematopoietic פרימיטיבי, מהווים הסופית מתאי גזע Pluripotent אנושי

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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.

רומן חדש פאט שומן טכניקת השתלת לדמיין את כלי השיט הדור של כלי הדם בתאי גזע אנדותל

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.

גישה סטנדרטית עבור multispecies טיהור תאים יונקים זכר יונקים על ידי דיסוציאציה רקמות מכנית זרימת cytometry

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.

היישום של RNAi חום-הלם-induced בביטוי פקטור שעתוק כדי לתכנת בתאי הנבט את הנוירונים C . elegans

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

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion תא בודד בהמחיה שלם דג זברה

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

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).

להמחיש את צומת ואת צלחת Notochordal Gastrulating עוברי העכבר באמצעות מיקרוסקופ אלקטרונים סריקה כל הר Immunofluorescence

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.

בדור חוץ גופית של Somite נגזרים מן האדם המושרה בתאי גזע Pluripotent

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