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

優雅、スクリーンで使用される shRNA ライブラリを提供するメルボルン大学のロス ディキンズに感謝いたします。この作業によって、レット症候群の研究を信頼 (a. b.) 資金を供給されました。

動物を含むすべての手順は、フレッド ・ ハッチンソンがん研究センター機関動物ケアおよび使用委員会 (IACUC) によって承認されたプロトコルを使用して行われました。5-アザシチジン (5 字) を除いて重大な健康リスクを引き起こす知られていますここで使用する試薬はありません。
1. Xi にMeCP2- リュック ・ HR 遺伝子レポーター細胞ラインを生成します。
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<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>

私たちの最近の研究6ルシフェラーゼとマウス セルラインを生成、hygromycin 抵抗性遺伝子、Xi にMeCP2を融合し、ライブラリを導入 > 60,000 Shrna ターゲット > 25,000 の遺伝子。我々 有利さを見つけて 30 遺伝子がノックダウン授与生存 hygromycin B 選択で、 MeCP2と x 染色体のサイレンシングの制御における役割を示唆しています。これらの結果はあった個々 のヘアピンで...

1 つ女性、レット症候群、遺伝性精神障害の最も一般的なフォームMeCP2、正常な神経機能1に不可欠なタンパク質をエンコードする x 染色体遺伝子のヘテロ変異が原因です。MeCP2表現の復元だったように病1,のマウスモデルで神経学的な欠損を逆に、この疾患の治療への潜在的なアプローチは、Xi に野生型のMeCP2遺伝子の再活性化になります。2,4しますただし、メスの細胞における染色体 X 2 つのエピジェネティックなサイレンシングはしっかりと生物3,4の場合は、生涯維持と Xi 遺伝子の強力な再活性化の主要な必要があります。複数のエピジェネティックな調節経路で中断。
MeCP2のサイレンシングのメンテナンスに必要な要因を識別するために我々 は最初にMeCP2- ルシフェラーゼ - hygromycin 抵抗遺伝子融合 (MeCP2 リュック HR) を乗せて X 染色体 2 つのトランスジェニック マウス モデルを開発 (XMeCP2-LUC-HR/XMeCP2)5。不安定で機能、(XMeCP2 リュック HR/Y) hemizygous 男性で模倣した表現型のMeCP2削除、レポーター遺伝子の表現の損失で起因したことが分かったMeCP2融合コンストラクトの表現が内因性 MeCP25の式と一致するパターンで容易に検出されました。非アクティブまたはアクティブのいずれかの x 染色体の構築と不死化線維芽細胞クローンを生成し、その元表現野生型 MeCP2 およびない記者の構築; 確認逆は、後者の本当だった。Xi (「レポーター細胞」) のレポーター細胞が作成を再アクティブ化することができることを示す生物発光アッセイの活動を得られる DNA 遺伝子サイレンシング廃止に知られているエージェントを demethylating 5-アザシチジン (5 字) に露出されたとき、したがって遺伝学的スクリーニングに使用されます。
次にMeCP2のサイレンシングのレギュレータのハイスループット遺伝学的スクリーニングを行った。記者のセルライン レトロ ウイルス ライブラリを含む感染した最初 > 60,000 異なる Shrna ターゲット >6マウスのゲノム5,の中の 25,000 の遺伝子 hygromycin B 選択を。ヘアピン周波数を事前に比較したし、次世代シーケンシングによる事後選択サンプル、記者として再活性化 hygromycin B 選択成長の優位性を与えられる責任のヘアピンの濃縮の結果します。このアプローチを使用して、我々 は 30 遺伝子サイレンシング、 MeCP2の関与し、個々 のヘアピンのレポーター細胞の伝達と、ルシフェラーゼ活性測定による調査結果を確認後。

<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

前方遺伝スクリーン、ヘテロクロマチンに挿入されたレポーター遺伝子を使用しては、モデル有機体のエピジェネティックな調節機構を調査するため広く使用されています。短いヘアピン Rna (Shrna) とクラスター化された空間定期的に短い回文繰り返し (CRISPR) などの技術は、二倍体の細胞でこのような画面を有効にしています。ここで説明の x 染色体の不活性化 (嘲りながら)、レギュレータの大規模 shRNA 画面マウス細胞株を用いたホタルのルシフェラーゼとメチル CpG 結合タンパク質の C 末端で hygromycin 抵抗性遺伝子ノックアウト 2 (MeCP2) 遺伝子非アクティブな x 染色体 (Xi)。レポーター細胞ラインの構造の再授与 hygromycin B 選択で、大きな shRNA ライブラリを選別し、ヘアピン、事後選択濃縮を使用して測定することによって、記者を再アクティブ化する私たちを有効に存続の利点次世代シーケンス。豊かなヘアピンが Xi のルシフェラーゼ レポーターを有効にする能力をテストすることによって個別に検証されます。

マウス腱線維芽細胞が収穫されたMeCP2 リュック HR/X X 上記からMeCP2雌マウス、HPV 16 virus7 から E6 と E7 遺伝子のレトロ ウイルスの伝達によって不死化制限の希釈を使用してクローンを作成およびテストルシフェラーゼ活性と野生型の MeCP2 タンパク質 (図 1 a および 1 b) の発現。ルシフェラーゼ活性は記者は、Xa、堅牢性と検出されない場合 Xi;?...

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

プールされた shRNA MeCP2 再アクティブ化非アクティブ X 染色体上の画面

小さいヘアピン RNA (shRNA) とホタルのルシフェラーゼとマウス細胞における x 染色体不活性化のレギュレータを識別するの次世代シーケンス ベースのプロトコルについて報告して、メチル CpG 結合蛋白質 2 (hygromycin 抵抗性遺伝子融合MeCP2)不活性 X 染色体の遺伝子。

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.

Research

JoVE Journal

Genetics

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

細菌接合に関与する転写タンパク質の配列特異的分析のための接合交配アッセイ

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

円の染色体構造の次世代シーケンシングによる遺伝子制御配列の高スループット識別キャプチャ (4 C 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

分裂酵母分裂酵母のディープ シーケンス アシスト、自発的な抑制画面

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

不活性 X 染色体上の Mecp2 の薬理学的再活性化のための非ランダムマウスモデル

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

非正規の開いたリーディング ・ フレームから翻訳された蛋白質を発表する OpenProt データベースを利用した質量分析を用いたプロテオミクス解析

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

哺乳類細胞におけるCRISPRベースの遺伝子スクリーンのプール

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

ショウジョウバエを用いたヘテロクロマチン促進薬の同定のためのスクリーニング方法

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

定量染色体立体構造捕捉による胚体におけるエンハンサー・プロモーター接触の同定(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

サッカロミセス・セレビシエにおける年代の寿命を調節する遺伝的リンクの特性評価のためのサプレッサースクリーン

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

D1S80ローカスを用いたDNAフィンガープリントのラボクラスへの応用

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