Name: pooled shrna screen for reactivation of mecp2 on the inactive x chromosome
Uploaded: 2018-03-02T13:00:00
Description: Watch this Scientific Journal Video about Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome at JoVE.com

Grazie Ross Dickins dell'Università di Melbourne per fornire gentilmente la libreria di shRNA utilizzata nella schermata. Questo lavoro è stato finanziato da Rett sindrome Research Trust (A.B.).

Tutti i passaggi che coinvolgono animali sono stati effettuati utilizzando protocolli approvati dal Fred Hutchinson Cancer Research Center istituzionale Animal Care e uso Committee (IACUC). Non utilizzare reagenti utilizzati qui sono noti rischi significativi per la salute umana ad eccezione di 5-azacitidina (5-AZA).
1. generazione di una linea cellulare reporter con MeCP2- LUC-HR transgene su Xi
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Preparazione dei fibroblasti del tendine del mouse da una donna X/x<su...

<ol>
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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=Rett+syndrome+is+caused+by+mutations+in+X-linked+MECP2,+encoding+methyl-CpG-binding+protein+2.">Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.</a> Nat Genet. 23, 185-188 (1999).</li><li>Guy, J., Gan, J., Selfridge, J., Cobb, S., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+neurological+defects+in+a+mouse+model+of+Rett+syndrome.">Reversal of neurological defects in a mouse model of Rett syndrome.</a> Science. 315, 1143-1147 (2007).</li><li>Maduro, C., de Hoon, B., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+the+Puzzle+Pieces:+the+Bigger+Picture+of+XCI.">Fitting the Puzzle Pieces: the Bigger Picture of XCI.</a> Trends Biochem Sci. 41, 138-147 (2016).</li><li>Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosage+compensation+of+the+sex+chromosomes+and+autosomes.">Dosage compensation of the sex chromosomes and autosomes.</a> Semin Cell Dev Biol. 56, 9-18 (2016).</li><li>Wang, J., 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=Wild-type+microglia+do+not+reverse+pathology+in+mouse+models+of+Rett+syndrome.">Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.</a> Nature. 521, E1-E4 (2015).</li><li>Sripathy, 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=Screen+for+reactivation+of+MeCP2+on+the+inactive+X+chromosome+identifies+the+BMP/TGF-beta+superfamily+as+a+regulator+of+XIST+expression.">Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-beta superfamily as a regulator of XIST expression.</a> Proc Natl Acad Sci U S A. 114, 1619-1624 (2017).</li><li>Foster, S. A., Galloway, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+papillomavirus+type+16+E7+alleviates+a+proliferation+block+in+early+passage+human+mammary+epithelial+cells.">Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells.</a> Oncogene. 12, 1773-1779 (1996).</li><li>Hnasko, T. S., Hnasko, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> Methods Mol Biol. 1318, 87-96 (2015).</li><li>Strezoska, Z., 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=Optimized+PCR+conditions+and+increased+shRNA+fold+representation+improve+reproducibility+of+pooled+shRNA+screens.">Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled shRNA screens.</a> PLoS One. 7, e42341 (2012).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+High-Molecular-Weight+DNA+from+Mammalian+Tissues+Using+Proteinase+K+and+Phenol.">Isolation of High-Molecular-Weight DNA from Mammalian Tissues Using Proteinase K and Phenol.</a> Cold Spring Harb Protoc. 2017, (2017).</li><li>Goto, Y., Takagi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraploid+embryos+rescue+embryonic+lethality+caused+by+an+additional+maternally+inherited+X+chromosome+in+the+mouse.">Tetraploid embryos rescue embryonic lethality caused by an additional maternally inherited X chromosome in the mouse.</a> Development. 125, 3353-3363 (1998).</li><li>Csankovszki, G., Nagy, A., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+of+Xist+RNA,+DNA+methylation,+and+histone+hypoacetylation+in+maintaining+X+chromosome+inactivation.">Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation.</a> J Cell Biol. 153, 773-784 (2001).</li><li>Minajigi, A., 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=Chromosomes.+A+comprehensive+Xist+interactome+reveals+cohesin+repulsion+and+an+RNA-directed+chromosome+conformation.">Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation.</a> Science. 349, (2015).</li><li>McHugh, C. A., 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+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521, 232-236 (2015).</li><li>Bhatnagar, 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=Genetic+and+pharmacological+reactivation+of+the+mammalian+inactive+X+chromosome.">Genetic and pharmacological reactivation of the mammalian inactive X chromosome.</a> Proc Natl Acad Sci U S A. 111, 12591-12598 (2014).</li><li>Minkovsky, A., 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+Mbd1-Atf7ip-Setdb1+pathway+contributes+to+the+maintenance+of+X+chromosome+inactivation.">The Mbd1-Atf7ip-Setdb1 pathway contributes to the maintenance of X chromosome inactivation.</a> Epigenetics Chromatin. 7, 12 (2014).</li><li>Minkovsky, A., 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+screen+of+inactive+X+chromosome+reactivation+identifies+the+enhancement+of+DNA+demethylation+by+5-aza-2'-dC+upon+inhibition+of+ribonucleotide+reductase.">A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2'-dC upon inhibition of ribonucleotide reductase.</a> Epigenetics Chromatin. 8, 42 (2015).</li></ol>

Nel nostro recente studio6, abbiamo generato una linea cellulare murina con luciferasi e geni di resistenza igromicina fusa per MeCP2 su Xi ed esso trasdotte con una libreria di > 60.000 shRNA targeting > 25.000 geni. Abbiamo trovato 30 geni cui vantaggio di sopravvivenza hanno conferito colpo sotto selezione igromicina B, suggerendo loro ruolo nel controllo di MeCP2 e silenziamento del x-cromosoma. Questi risultati sono stati convalidati trasdurre la linea cellulare segnalati co...

Uno le forme più comuni di danno mentale ereditario nelle femmine, la sindrome di Rett, è causato da mutazioni eterozigoti in MeCP2, un gene del cromosoma x che codifica per una proteina essenziale per la normale funzione di un neurone1. Un potenziale approccio per trattare questo disordine sarebbe riattivazione del selvaggio-tipo allele di MeCP2 su Xi, come ripristino dell'espressione di MeCP2 è stata indicata per invertire i deficit neurologici in un modello murino di questa malattia1, 2 , 4. Tuttavia, silenziamento epigenetico di uno dei due cromosomi in cellule femminili X sia strettamente mantenuto durante tutta la durata della vita di un organismo3,4, e robusta riattivazione di un gene di Xi probabilmente richiederebbe una maggiore rottura in più vie di regolazione epigenetiche.
Per identificare i fattori necessari per la manutenzione del silenziamento di MeCP2 , abbiamo sviluppato un modello di topo transgenico che trasportano una fusione del gene MeCP2- luciferasi - igromicina resistenza (MeCP2-LUC-HR) su uno dei due cromosomi X (X MeCP2-LUC-HR/xMeCP2)5. Sebbene la MeCP2 espresso dal costrutto fusione ha dimostrato di essere instabile ed ha provocato una perdita di funzione, fenotipico che imita l'eliminazione MeCP2 nei maschi emizigoti (X/y diMeCP2-LUC-HR), l'espressione dei geni reporter era prontamente rilevabile in un modello costante con l'espressione endogena di MeCP25. Abbiamo quindi generato cloni dei fibroblasti immortalati con il costrutto sul cromosoma x attivo o inattivo e ha confermato che l'ex espresso wild type MeCP2 e non la costruzione del reporter; l'inverso è vero per quest'ultimo. Quando esposto ad un DNA demethylating agente conosciuto per abrogare il silenziamento genico, 5-azacitidina (5-AZA), cellule con il reporter su Xi ("cellule di reporter") guadagnato attività in un'analisi di bioluminescenza, che indica che il nostro costrutto potrebbe essere riattivato e quindi utilizzato per lo screening genetico.
Abbiamo sviluppato successivamente una schermata genetica ad alta produttività per regolatori del silenziamento di MeCP2 . La linea cellulare reporter in primo luogo è stata infettata con una libreria retrovirale contenenti > 60.000 diversi shRNA targeting > 25.000 geni in tutto il genoma del mouse5,6e quindi sottoposti a selezione igromicina B. Frequenza tornante è stata confrontata in pre- e post-selezione campioni mediante sequenziamento di nuova generazione, come reporter riattivazione conferiti vantaggio di crescita sotto igromicina B selezione e arricchimento di forcine responsabile ha provocato. Utilizzando questo approccio, abbiamo identificato 30 geni implicati in MeCP2 silenziamento e successivamente ha confermato i risultati transducing le cellule reporter con forcine individuali e misurando la loro attività luciferasica.

<table>
	<tbody>
		<tr>
			<td>293FT Cell Line</td>
			<td>Invitrogen</td>
			<td>R700-07</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Azacytidine</td>
			<td>Sigma</td>
			<td>A2385-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP</td>
			<td>Beckman-Coulter</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MECP2 antibody</td>
			<td>Millipore</td>
			<td>07-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C2674-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96-Well Solid White Polystyrene Microplates</td>
			<td>Fisher Scientific</td>
			<td>07-200-336</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Expression Arrest microRNA-adapted Retroviral Vector (pMSCV)</td>
			<td>Open Biosystems</td>
			<td>EAV4679</td>
			<td></td>
		</tr>
		<tr>
			<td>Expression Arrest pSM2 Retroviral shRNAmir library</td>
			<td>Open Biosystems</td>
			<td>RMM3796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisherbrand</td>
			<td>03-600-511</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2500</td>
			<td>Illumina</td>
			<td>SY&#8211;401&#8211;2501</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Calbiochem</td>
			<td>400051-1MU</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Glo Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2610</td>
			<td>Homogenous Assay for Screening Colonies</td>
		</tr>
		<tr>
			<td>Luciferase Assay System</td>
			<td>Promega</td>
			<td>E4530</td>
			<td>Non-Homogenous Assay for Testing Individual Hairpins</td>
		</tr>
		<tr>
			<td>Luminometer TopCount NXT</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
			<td>Or similar luminometer</td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v2</td>
			<td>Illumina</td>
			<td>MS-102-2001</td>
			<td>Use kit compatible with your equipment</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>#12259</td>
			<td>VSV-G envelope vector</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>#12260</td>
			<td>Packaging vector</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Gibco</td>
			<td>A11138-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
	</tbody>
</table>

pooled shrna screen for reactivation of mecp2 on the inactive x chromosome

Avanti schermi genetici usando geni reporter inseriti l'eterocromatina sono stati ampiamente utilizzati per studiare i meccanismi di controllo epigenetico in organismi modello. Tecnologie tra cui short hairpin RNA (shRNA) e cluster regolarmente intercapedine ripetizioni brevi palindromi (CRISPR) hanno permesso tali schermi in cellule di mammifero diploide. Qui descriviamo una schermata di shRNA su larga scala per regolatori di inattivazione del cromosoma x (XCI), utilizzando una linea cellulare murina con luciferasi firefly e geni di resistenza igromicina bussò in al C-terminale di methyl CpG binding protein al 2 (MeCP2) gene sul cromosoma x inattivo (Xi). Riattivazione del costrutto nella linea cellulare reporter conferiti vantaggio di sopravvivenza sotto selezione igromicina B, consentendoci di schermo una biblioteca grande shRNA e identificare forcine che riattivato il reporter misurando il loro utilizzo di arricchimento post-selezione sequenziamento di nuova generazione. Le forcine arricchite poi sono state convalidate individualmente verificando la loro capacità di attivare il reporter di luciferase su Xi.

Fibroblasti di topo del tendine sono state raccolte da un precedentemente descritto X/xMeCP2-LUC-HRmouse femminileMeCP2 , immortalata da trasduzione retrovirale degli oncogeni E6 ed E7 di HPV-16 virus7, clonate mediante diluizione limitante e testato per l'attività luciferasica ed espressione della proteina MeCP2 selvaggio-tipo (Figura 1A e 1B). L'attività luciferasica era robusto, se il giornalista era XA e non rilevabile se il Xi; l'espressione di M...

Watch this Scientific Journal Video about Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome at JoVE.com

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

In pool shRNA schermo per riattivazione di MeCP2 sul cromosoma di X inattivo

Segnaliamo un short hairpin RNA (shRNA) e sequenziamento basato su protocollo di ultima generazione per l'identificazione dei regolatori di inattivazione del cromosoma x in una linea cellulare murina con luciferasi firefly e geni di resistenza igromicina fuse con il metil CpG binding protein 2 ( MeCP2) gene sul cromosoma di X inattivo.

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

Begin this procedure with preparation of mouse tendon fibroblasts, as described in the text protocol. Harvest the cells from a 10-centimeter plate by first aspirating the media. Then add one milliliter of 0.05%trypsin EDTA and incubate for two to five minutes at 37-degrees Celsius.Following incubation, quench the trypsin with nine milliliters of DMEM-10, and collect the resuspended cells in a 15-milliliter conical tube. Dilute the cells with DMEM-10 to a final concentration of one cell per 100 microliters. Then transfer the cells to 96 well plates by pipetting 100 microliters of the suspension into each well.Incubate the cells at 37 degrees Celsius until the wells are confluent, which can take up to two to three weeks. Replace the media with fresh DMEM-10 every three to five days. When confluent, mobilize the cells with 20 microliters 0.05%trypsin EDTA per well, and split them into two duplicate sets of 96 well plates.Assay for luciferase activity in one set of plates. Use a homogenous Firefly luciferase assay that does not require media removal, following the protocol included with the luciferase assay kit. Use the assay results to select clones with no detectable luciferase activity from the remaining set of plates.Expand these clones first to a 24 well plate, and then to a six well plate. Perform the same for a clone with the highest luciferase activity. This will be used as a positive control for validation purposes.Following expansion in the six well plate, harvest an aliquot of each well for a Western blot. Mobilize the cells with 200 microliters of 0.05%trypsin EDTA. Incubate the cells for two to five minutes at 37 degrees Celsius.Quench the cells with two milliliters of DMEM-10, and transfer half of the cells to another six well plate. Wait until the cells attach to the bottom of the plate. Then wash the cells once with PBS before the lysing for the Western blot.Plate several negative clones at 0.2 million cells per well in a six well plate. Treat the cells once with 10 micromolar 5-azacytidine. Then incubate for 72 hours without changing the media before assaying for luciferase activity, as before.Next, choose one negative clone that gained luciferase activity with 5-azacytidine for the screen, and begin expanding it to multiple 10-centimeter plates. Freeze the remaining negative clones in liquid nitrogen as a backup. The positive clone can be maintained in the tissue culture or frozen until further use.In the morning, plate one million inactive X cells onto 10-centimeter tissue culture plates. In the afternoon, infect the plates by replacing the media with fresh DMEM-10 containing a predetermined amount of viral supernatant and five to 10 micrograms per milliliter of polybrene. After 18 to 24 hours, aspirate the media and add 10 milliliters of fresh DMEM-10 to each plate.Culture plates for another 48 hours. In the morning, mobilize the cells using one milliliter of 0.05%trypsin EDTA per plate, and quench the cells with 9 milliliters of DMEM-10 per plate. Run the suspension through a 100-micron mesh filter.Pull the filtered suspensions together, and seed two sets of 10-centimeter plates at one million cells per plate. In the afternoon, replace the media in one set of plates with DMEM-10 containing 15 micrograms per milliliter of Hygromycin B.In the other set, replace the media with fresh DMEM-10 only. After 48 hours, mobilize the cells from Hygromycin B untreated plates using one milliliter of 0.05%trypsin EDTA.Incubate for two to five minutes at 37 degrees Celsius. Next, quench the cells with 9 milliliters of DMEM-10. Pull the individual samples, and proceed immediately with extracting DNA.Continue culturing the Hygromycin B selection set for six more days, replenishing the selection media with fresh DMEM-10 containing Hygromycin B every other day. On day six, replace the selection media with fresh DMEM-10 only. Allow four days for recovery, and then harvest the post-selection sample DNA as before.Proceed to identify hairpins enriched in the post-selection samples, as described in the text protocol. To validate the hits, first seed eight million 293-FT cells per plate on 10-centimeter plates. The following day, replace the media with nine milliliters of antibiotic-free DMEM-10 per plate 30 minutes before transection.Next, add 0.5 milliliters of transfection mix one to each aliquot of transfection mix two, and gently mix by pipetting. Then incubate the mixture at room temperature for 20 minutes. Apply the mixture onto each plate in a drop-wise fashion, and swirl the plate gently to mix.The following day, replace the media with five milliliters of fresh DMEM-10.48 hours after initial transfection, collect the lentiviral supernatant, and run it through a 0.22-micron filter that has been prewet with DMEM-10 to maximize recovery. The next morning, plate 0.1 million inactive X cells per well onto six well plates. In the afternoon, infect the plates by replacing the media with two milliliters of fresh DMEM-10 containing lentiviral supernatant and five to 10 micrograms per milliliter of polybrene.Infect a control well with viral supernatant produced from an empty lentiviral vector. The following day, replace the media with two milliliters of DMEM-10 containing one milligram per milliliter of puromycin. Use selecting for four days.After four days, replace the media with two milliliters of fresh DMEM-10, and allow 48 to 72 hours for recovery. After mobilizing the cells with 150 microliters of 0.05%trypsin EDTA, incubate them for two to five minutes at 37 degrees Celsius. Quench the cells with 1.5 milliliters of DMEM-10 before transferring to conical tubes and centrifuging the cells at room temperature at 500 times G for 10 minutes.Remove the media and resuspended the cells in 100 microliters of lysis buffer provided in the luciferase assay kit. Incubate the suspension at room temperature for five minutes. After five minutes, transfer the suspension to a white 96 well plate.Perform the assay in the darkroom, following the protocol included in the kit. Use active X cells as a positive control and negative X cells infected with an empty lentiviral vector as a negative control. Immortalized mouse tendon fibroblast clones show either positive or negative Firefly luciferase activity, consistent with the placement of the reporter on the active or inactive X chromosome, respectively.MeCP2 expression, as determined by the Western blot, exhibits the reciprocal pattern. Note that Firefly luciferase activity can be induced in negative clones with administration of 5-azacitidine. If present on the active X chromosome, the Hygromycin resistance gene confers a survival advantage under Hygromycin selection, when compared to the cells with the gene on the inactive X chromosome.After watching this video, you should have a good understanding of how to design and perform a screen for regulators of X-chromosome inactivation using reporters selectively inserted on inactive X chromosome. My laboratory has previously carried out reported base screens for chromatin regulators in yeast, and our success with this approach inspired me to design this screen for regulators of X-chromosome inactivation in mammalian cells. Following this procedure, other methods, such as using small, interfering RNAs, CRISPR, small molecules can be performed to help answer additional questions regarding X-chromosome inactivation in mammalian cells.

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Una schermata di soppressore Deep-sequenziamento-assistita, spontanea nel lievito a fissione Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Un modello di mouse non casuale per la riattivazione farmacologica di MECP2 sul cromosoma X inattivo

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Basati sulla spettrometria di massa proteomica analisi utilizzando il Database di OpenProt a svelare nuove proteine tradotte da Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Schermi genetici basati su CRISPR in cellule mammaliane

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Un metodo di screening per l'identificazione di farmaci che promuovono l'eterocromatina utilizzando la drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

Identificazione dei contatti Enhancer-Promoter nei corpi embrionali mediante Quantitative Chromosoma Conformation Capture (4C)

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

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

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

Uno schermo soppressore per la caratterizzazione dei collegamenti genetici che regolano la durata cronologica in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Applicazione del rilevamento delle impronte digitali del DNA utilizzando il locus D1S80 nelle classi di laboratorio

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...