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

Ross Dickins Melbourne Üniversitesi nezaketle belgili tanımlık perde içinde kullanılan shRNA Kütüphane sağlamak için teşekkür ederiz. Bu eser Rett Sendromu araştırma güven (A.B. tarafından) finanse edildi.

Hayvanları içeren tüm adımları Fred Hutchinson Kanser Araştırma Merkezi Kurumsal hayvan bakım ve kullanım Komitesi (IACUC) tarafından onaylanmış protokolleri kullanılarak yapılmıştır. Burada kullanılan hiçbir reaktifler 5-azacytidine (5-AZA) dışında önemli insan sağlığı risk teşkil olduğu bilinmektedir.
1. bir muhabiri hücre satırıyla MeCP2- LUC-HR transgene Xi üzerinde oluşturma
<ol><li>
Gelen kadınMeCP2-LUC-HR /X X fare tendon ...

<ol>
	<li>Amir, R. 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>

Bizim son6çalışma, biz bir fare hücre kültürünü luciferase ile oluşturulan ve hygromycin direnç genleri MeCP2 Xi üzerinde erimiş ve bir kütüphane ile transduced > shRNAs 60.000 hedefleme > 25.000 gen. 30 genler olan knock-down haiz sağkalım avantajı hygromycin B seçimi altında MeCP2 ve x kromozomu susturmak kontrol içindeki rollerine düşündüren bulduk. Bu sonuçlar bireysel saç tokaları ile bildirilen hücre kültürünü transducing ve sessiz luciferase...

Bir kadın, Rett sendromu, kalıtsal akıl bozukluğu en yaygın formları be neden yanında MeCP2, normal nöronal fonksiyon1için önemli bir protein kodlar bir x kromozomu gen heterozigoz mutasyonlar. Bu hastalık1, , bir fare modeli nörolojik açıkları tersine çevirmek için restorasyon MeCP2 ifadesinin gösterildiği bu hastalığın tedavisi için potansiyel bir yaklaşım vahşi tipi MeCP2 alleli Xi üzerinde reaktivasyonu olacaktır 2 , 4. ancak, bir iki X kromozomları dişi hücrelerdeki epigenetik susturmak sıkı bir organizma3,4ömrü muhafaza ve sağlam bir Xi gen reaktivasyonu büyük olasılıkla büyük bir gerektirir birden çok epigenetik yasal yolları bozulma.
Biz faktörler MeCP2 damping bakım için gerekli belirlemek için önce MeCP2- luciferase - hygromycin direnç gen füzyon (MeCP2-LUC-HR) birinde iki X kromozomu taşıyan bir transgenik fare modeli geliştirildi (X MeCP2-LUC-HR/XMeCP2)5. Her ne kadar füzyon yapı ifade MeCP2 kararsız ve işlevi, phenotypically taklit eden MeCP2 silme (XMeCP2-LUC-HR/y) hemizygous erkeklerde, muhabir gen ifade kaybına neden olduğu ortaya çıktı kolayca algılanabilir bir desen endojen MeCP25ifadesi ile tutarlı oldu. Daha sonra ölümsüzleştirdi fibroblast klonlar etkin olmayan veya active x kromozomu üzerinde yapı ile oluşturulan ve o eski vahşi türü MeCP2 ve değil muhabiri yapı ifade doğruladı; tersini ikincisi için doğruydu. Gen susturmak, iptal etmek için bilinen Ajan demethylating bir DNA için 5-azacytidine (5-AZA), maruz kaldığında Xi ("muhabiri hücre") muhabiri hücre bizim yapı yeniden gösteren bir bioluminescence tahlil etkinliği kazandı ve bu nedenle genetik tarama için kullanılan.
Biz sonraki yüksek üretilen iş genetik bir ekran MeCP2 damping düzenleyiciler için geliştirilmiştir. Muhabiri hücre kültürünü ilk içeren bir retroviral kütüphane ile enfekte > 60.000 farklı shRNAs hedefleme > fare genom5,6, boyunca 25.000 genler ve sonra hygromycin B seçime tabi. Saç tokası frekans içinde ön karşılaştırıldı ve sonraki nesil sıralama kullanarak sonrası seçim örnekleri, muhabir olarak yeniden etkinleştirme büyüme avantajı hygromycin B seçimi altında doğan ve sorumlu saç tokaları zenginleştirme sonuçlandı. Bu yaklaşımı kullanarak, biz 30 genler susturmak MeCP2 içinde karıştığı tespit ve sonra bulgular muhabiri hücre bireysel saç tokaları ile transducing ve luciferase faaliyetlerini ölçme doğruladı.

<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

Heterochromatin eklenen reporter genler kullanarak ileri genetik ekranlar kapsamlı model organizmalar epigenetik kontrol mekanizmaları araştırmak için kullanılmaktadır. Kısa saç tokası RNA'ların (shRNAs) ve kümelenmiş düzenli olarak interspaced kısa palindromik yineler (CRISPR) gibi teknolojiler böyle ekranlar diploit memeli hücrelerinde etkinleştirdiniz. Burada bir fare hücre satırı ile ateş böceği luciferase kullanarak x kromozomu inactivation (XCI), düzenleyiciler için bir büyük ölçekli shRNA ekran açıklamak ve hygromycin direnç genleri içinde metil CpG bağlayıcı protein C-terminus adlı 2 (MeCP2) gen çaldı etkin olmayan x kromozomu üzerinde (XI). Muhabiri hücre kültürünü inşa reaktivasyonu sağkalım avantajı hygromycin B seçimi, büyük shRNA kitaplık ekranı ve onların sonrası seçim zenginleştirme kullanarak ölçerek muhabir yeniden saç tokaları tanımlamak olanaklı kılar altında doğan yeni nesil sıralama. Zenginleştirilmiş saç tokaları sonra tek tek XI luciferase kahramanlarına etkinleştirmek için yeteneklerini sınayarak geçerliliği.

Fare tendon fibroblastlar hasat--dan bir daha önce açıklananMeCP2-LUC-HR/X XMeCP2 dişi fare, E6 ve E7 oncogenes HPV-16 virus7, üzerinden retroviral iletim tarafından ölümsüzleştirilmiş sınırlayıcı dilüsyonu kullanılarak kopyalanmış ve test luciferase etkinliği ve ifade vahşi tipi MeCP2 protein (şekil 1A ve 1B). Luciferase aktivite sağlam muhabir Xa üzerinde olsaydı ve Xi Eğer üzerinde tespit edilemez; Yerel MeCP2 ifade karşı...

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

Havuza alınan shRNA MeCP2 reaktivasyonu üzerinde etkin olmayan X kromozomu için ekran

Biz bir küçük saç tokası RNA (shRNA) ve düzenleyiciler, x kromozomu inactivation ateş böceği luciferase ile bir fare hücre satırı tanımlamak için sonraki nesil sıralama tabanlı iletişim kuralı raporu ve hygromycin direnç genleri metil için erimiş CpG bağlayıcı protein 2) MeCP2) etkin olmayan X kromozomu üzerindeki gen.

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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Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Bakteriyel Konjugasyon ilgilendim Transferi Proteinlerin Dizi özgü Analiz konjugatif Çiftleşme Tahliller

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Gen düzenleyici dizileri dairesel kromozom Conformation yeni nesil sıralama kullanarak yüksek üretilen iş tanımlaması yakalama (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Fisyon mayası Schizosaccharomyces pombe derin sıralama yoluyla, kendiliğinden bastırıcı ekran

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

Pasif X kromozom üzerinde Mecp2 farmakolojik yeniden aktivasyonu için rasgele olmayan bir fare modeli

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

Kütle spektrometresi tabanlı proteomik analizleri roman proteinler açıklayacak OpenProt veritabanı kullanarak kanonik olmayan açık okuma çerçevesi tercüme

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

Memeli Hücrelerinde Havuzlu CRISPR Tabanlı Genetik Ekranlar

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

Drosophila Kullanan Heterokromatin-Teşvik Edici İlaçların Tanımlanması nda Bir Tarama Yöntemi

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

Kantitatif Kromozom Konformasyon Yakalama (4C) ile Embriyoid Cisimlerde Arttırıcı-Organizatör Kontalarının Belirlenmesi

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

Saccharomyces cerevisiae Kronolojik Yaşam Süresini Düzenleyen Genetik Bağlantıların Karakterizasyonu için Bastırıcı Ekran

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

Laboratuvar Sınıflarında D1S80 Locus Kullanılarak DNA Parmak İzi Uygulaması

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