Name: pooled shrna screen for reactivation of mecp2 on the inactive x chromosome
Uploaded: 2018-03-02T13:00:00.000+00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome at JoVE.com

我们感谢墨尔本大学的罗斯 Dickins 慷慨地提供了 shRNA 图书馆在屏幕上使用。这项工作由 Rett 综合症研究信托基金资助。

所有涉及动物的步骤都是使用弗雷德？哈钦森癌症研究中心机构动物保育和使用委员会 (IACUC) 批准的协议进行的。除了 5-azacytidine (5-AZA) 以外, 这里所用的试剂都不知道对人体健康有重大危害。
1. 用MeCP2-卢克-HR 转基因在西安生成一个记者细胞线
<ol><li>
从雌性 xMeCP2-LUC-HR /XMeCP2鼠标中制备小鼠肌腱成纤维细胞
	<ol>
<li>弄死10-12 天大的女性 XMeCP2-LUC-HR/XMeCP2鼠标。</li>
		<li>使用直解剖剪刀, 做一个圆周切口通过皮肤的近尾部部分。</li>
		<li>把尸体靠近尾部的底部, 从老鼠身上拉出皮肤, 露出尾部的 fibrocartilaginous 部分。使用手术手术刀将暴露组织的10毫米的末端切除并切割成1毫米长的碎片。</li>
		<li>将零碎的组织转移到含有5毫升 Dulbecco 的改良鹰培养基 (DMEM) 的15毫升圆锥管中, 辅以1的 U/毫升青霉素, 1 µg/毫升链霉素, 1.5 毫克/毫升胶原酶, 预过滤通过0.45 µm, 然后0.2 µm 网状过滤器。连续旋转, 在37摄氏度孵育3-4 小时。</li>
		<li>将样品在室温下离心 500 x g , DMEM 10 毫升的并用重悬颗粒, 辅以10% 胎牛血清、1 U/毫升青霉素、1µg/毫升链霉素 (DMEM-10) 和6井组织培养板上的板。通过细胞一次, 并扩大到一个10厘米板块, 然后继续下一步。</li>
		<li>不朽细胞通过执行传导与表达载体携带 HPV-16 病毒癌基因 (E6/E7), 如前所述7。</li>
	</ol>
</li>
	<li>
选择单个单元格克隆并表征MeCP2报告器表达式
	<ol>
<li>从10厘米厚的盘子里收割细胞。首先, 吸入培养基, 加入1毫升0.05% 胰蛋白酶-EDTA 和孵育2-5 分钟在37摄氏度。用9毫升的 DMEM-10 淬火, 在15毫升圆锥管中收集悬浮细胞。</li>
		<li>用 DMEM-10 稀释细胞, 最终浓度为 1 cell/100 µL. 通过吹打100µL 将悬浮液输送到每个井中, 将其转移到96井板上。</li>
		<li>孵化37摄氏度, 直到水井汇合, 这可能需要长达2-3 周。每3-5 天更换一次新鲜 DMEM-10 的媒体。当汇合时, 动员细胞与20µL 0.05% 胰蛋白酶-EDTA 每井并且分裂他们成二个重复的套96井板材。</li>
		<li>用一组钢板测定荧光素酶的活性。使用均匀的萤火虫荧光素酶检测试剂盒, 不需要去除介质。按照套件附带的协议进行。</li>
		<li>使用步骤1.2.4 中的结果从剩余的板组中选择没有检测到的荧光素酶活性的克隆。将这些克隆扩展到24井, 然后再展开6井板。</li>
		<li>对具有最高荧光素酶活性的克隆执行相同的操作;这将用作验证目的的正则控制。</li>
		<li>在6井的板块中收获一整除, 为西方的印迹。动员细胞与200µL 0.05% 胰蛋白酶-EDTA。孵育2-5 分钟, 在37摄氏度。用2毫升的 DMEM-10 淬火, 将一半的细胞转移到1.5ml 离心管。在株溶藻前用 PBS 清洗细胞。或者, 将一半的细胞转移到另一个6井板上。当细胞附着在盘子的底部时, 用 PBS 和溶解冲洗出印迹。</li>
		<li>用 anti-MeCP2 抗体进行西方印迹, 确认荧光素酶阴性细胞表达, 而荧光素酶阳性细胞不表达活性 X 染色体上的野型 MeCP2;按照前面描述的协议8。使用任何野生类型的老鼠的脑组织裂解液作为一个积极的控制。</li>
		<li>在6井板中, 在 2 x 105单元格/井中, 板数个负克隆。治疗一次与10µM 5 AZA, 孵化72小时不改变培养基, 并化验荧光素酶活性, 如步骤1.2.3 所述。</li>
		<li>选择一个阴性克隆 (Xi 克隆), 获得荧光素酶活性与 5 AZA, 并开始扩大到多个10厘米板。将液氮中剩余的负克隆冻结为备用。阳性克隆 (Xa 克隆) 可以保持在组织培养或冷冻, 直到进一步使用。</li>
	</ol>
</li>
</ol>2. 使用潮霉素 B 选择对库进行重新激活的筛选
<ol><li>
感染测试克隆, 应用选择, 采集 DNA 进行测序
	<ol>
<li>获得病毒浓缩物的逆转录病毒构造表达发夹从一个 shRNA 图书馆, 或生产浓缩病毒上清, 使用所包含的协议所需的库。</li>
		<li>在继续使用屏幕之前, 滴定库 (如果可用, 则使用 GFP 标记)。目的为传染多样性在0.3 和0.5 之间。 注: 至少执行四个独立屏幕, 以尽量减少假阳性率。每屏使用20个板, 以获得100倍的库覆盖率, 以保持发夹的适当表示, 并避免随机辍学9。然而, 考虑到积极的选择屏幕和较大的库大小, 更低的覆盖率很可能就足够了。</li>
		<li>上午, 板 1 x 106 Xi 细胞到 10 cm 组织培养板材。</li>
		<li>下午, 通过更换含有病毒上清的新鲜 DMEM-10 和5-10 µg/毫升的凝聚胺来感染盘子。使用步骤2.1.2 中确定的病毒上清液量。</li>
		<li>18-24 小时后, 吸入介质, 并添加10毫升新鲜 DMEM-10 到每个板块。文化为另外 48 h。</li>
		<li>早晨, 用1毫升的0.05% 胰蛋白酶-EDTA 每板和淬火每板9毫升 DMEM-10 细胞。通过100µm 网格过滤器运行悬架。将过滤后的悬浮池组合在一起, 并在 1 x 106单元格/板上种子两组10厘米的板。</li>
		<li>下午, 在一组盘子中更换介质, DMEM-10 含有15µg/毫升的潮霉素 B (0 天)。在另一组中, 仅用新鲜 DMEM-10 替换介质。</li>
		<li>48小时后, 用1毫升0.05% 胰蛋白酶-EDTA 将细胞从未经处理的钢板中动员出来。孵育2-5 分钟, 在37摄氏度。用9毫升的 DMEM-10 淬火。将细胞转移到15毫升圆锥管和离心机在 500 x g 5 分钟室温。吸入上清液并立即进行, 从颗粒中提取 DNA。或者, 在离心步骤之前将细胞放入较大的管中。</li>
		<li>利用苯酚和 chroroform 的混合物提取 dna, 用醋酸钠和冷乙醇沉淀 dna, 如前所述10。池的个人预选 DNA 样品和存储在-20 °c, 直到进一步使用。</li>
		<li>继续培养潮霉素 B 选择集4天。在2天后, 用含有潮霉素 B 的新鲜 DMEM-10 补充选择介质。</li>
		<li>在7天, 只用新鲜的 DMEM-10 替换选择介质。允许2到4天的恢复, 然后按照步骤2.1.8 中的描述来收获细胞。按照步骤2.1.9 中的描述进行 DNA 提取。</li>
	</ol>
</li>
	<li>
在选择后的样品中识别出的发夹
	<ol>
<li>使用 5 ' 上下文 (5 '-ATCGTTGCCTGCACATCTTGG 3 ') 和 3 ' 反向环引物 (5 '-ACATCTGTGGCTTCACTA-3 '), 以放大半发夹从预选和后选择 DNA 样本。使用所有提取的 dna 在不同的 PCR 反应与 100-200 ng dna 每反应。 注: 这 5 ' 上下文底漆和 3 ' 反向环引物对应于 miR-30 上下文序列上游的 shRNA 插入和序列在 shRNA 循环, 分别。不同的库将需要不同的寡核苷酸。根据需要调整净化步骤以隔离不同的产品尺寸。</li>
		<li>使用尺寸选择珠去除引物和基因组 DNA, 并恢复100和 200 bp 的 PCR 产品. 按照珠子附带的协议。</li>
		<li>准备一个测序库来序列半发夹。每屏至少生成5000万个读取。按照适合于排序工具包和设备的协议进行。 注: 5000万读取每屏幕提供1000倍的表示, 每个 shRNA 在图书馆和大约10倍覆盖每一个独立的感染 (执行在100倍的代表, 每 shRNA), 确保充分的代表性每个感染事件。</li>
		<li>使用 Perl 脚本枚举每个 shRNA, 它用完美匹配来计算序列。选择在预选池中至少有10个读数的 shRNAs 进行进一步分析。根据1000年的置换计算充实作为后预选计数的比率, 并确定假发现率 (罗斯福)。选择的基因 shRNAs 与 < 5% 罗斯福作为 "命中" 进一步测试。</li>
	</ol>
</li>
</ol>3. 验证已识别的发夹 ("命中")
<ol><li>
包装慢病毒包含在屏幕上标识的 shRNAs
	<ol>
<li>种子 8 x 106 293FT 单元格每板在 10 cm 板材。</li>
		<li>第二天, 用9毫升的无抗生素 DMEM-10 每片更换培养基;在转染前30分钟进行。</li>
		<li>在30分钟间隔内, 准备两个混合用于转染:<ol>
<li>准备混合 1 (每板/shRNA; 根据 shRNAs 的总数扩大):10 µL 2 毫克/毫升聚乙烯亚胺 (裴) 在0.5 毫升的最佳最低基本介质。吹打混合, 室温孵育5分钟。</li>
			<li>准备混合 2 (每 shRNA): 4 µg 的 shRNA 向量被封装, 1.5 µg 的 VSV-g 信封向量 (pMD2) 和2.5 µg 的包装载体 (psPAX2), 混合在0.5 毫升的最佳最低基本介质。 注: 在包装中包括一个空的慢病毒载体向量, 用于以后的感染作为阴性控制。</li>
		</ol>
</li>
		<li>加入0.5 毫升混合1到每整除混合 2, 并轻轻地混合吹打。室温下在黑暗中孵化20分钟。</li>
		<li>用滴状的方式将混合物从步骤3.1.2 上涂抹到每个盘子上;轻轻地将盘子搅拌。</li>
		<li>第二天, 用5毫升的新鲜 DMEM-10 取代媒体。</li>
		<li>48小时后, 首次转染, 收集上清, 并通过一个0.22 µm 过滤器运行。预湿过滤器与 DMEM-10, 以最大限度地恢复。</li>
		<li>将过滤后的上清液整除为感染6井板的理想量。</li>
		<li>冷冻整除数在-80 摄氏度以后使用, 或立即进行转导。</li>
	</ol>
</li>
	<li>
荧光素酶的转导和试验研究 MeCP2 野生型基因在西安的活化和活化。
	<ol>
<li>上午, 板 1 x 105 Xi 单元格/井到6井板。</li>
		<li>下午, 用2毫升含有慢病毒载体上清的新鲜 DMEM-10 和5-10 µg/毫升的凝聚胺来取代培养基, 从而感染板块。通过一个空的慢病毒载体载体产生的病毒上清液感染控制良好。</li>
		<li>第二天, 用含有1毫克/毫升嘌呤霉素的2毫升 DMEM-10 取代培养基。文化四天。</li>
		<li>用2毫升新鲜 DMEM-10 替换培养基, 并允许48-72 小时恢复。</li>
		<li>动员细胞与150µL 0.05% 胰蛋白酶-EDTA。孵育2-5 分钟, 在37摄氏度。用1.5 毫升的 DMEM-10 淬火。将细胞转移到15毫升圆锥管和离心机在 500 x g在室温下10分钟。</li>
		<li>取出介质并并用重悬在荧光素酶检测试剂盒中提供的100µL 裂解缓冲液中的细胞。在室温下孵育5分钟。使用套件附带的协议将裂解物转移到白色96井板上, 并对荧光素酶活性进行检测。以 Xa 细胞为阳性对照, 并以慢病毒载体为阴性对照的 Xi 细胞感染。 注: 在化验前避免将白板暴露在荧光灯上;这将减少背景和增加化验灵敏度。如果可能的话, 检测试剂盒中提供的议定书被认为是最有效的。</li>
		<li>为了确定发夹是否对活性 X 染色体上的 MeCP2 表达水平有影响, 从 tranduced Xi 细胞中分离 RNA, 并采用定量 PCR 方法测定 MeCP2 mRNA 水平, 并将其用于扩增野生型 MeCP2 (F:5 '-TTCCATGCCAAGGCCAAACAG-3 ', R: 5 '-CCCATAAGGAGAAGAGACAACAGC-3 ')。</li>
		<li>
可选:在细胞从嘌呤霉素选择中恢复后, 增加 5-AZA 到最终浓度0.2 µM, 提高测定的灵敏度。在不替换培养基的情况下培养细胞72小时, 然后使用商业荧光素酶检测试剂盒检测荧光素素活性。</li>
		<li>测量沉默野生型 MeCP2 的恢复。在3.2.1 的协议步骤之后, 感染了 MeCP2 在 Xi 上的野生类型的 Xa 细胞, 含有单个发夹的载体。-3.2.4。将 RNA 从细胞中分离出来, 并用引物进行定量 PCR 测量 MeCP2 mRNA 水平, 目的是只放大野生型 MeCP2 (F: 5 '-TTCCATGCCAAGGCCAAACAG-3 ', R: 5 '-CCCATAAGGAGAAGAGACAACAGC-3 ')。</li>
	</ol>
</li>
</ol>

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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>

莱科博士作为弗雷德？哈钦森癌症研究中心的一名雇员对这篇文章做出了贡献。 所表达的观点是他自己的, 不一定代表国家卫生研究院或美国政府的意见。

在我们最近的研究6, 我们产生了一个小鼠细胞系与荧光素酶和潮霉素抵抗基因融合到MeCP2 上的 Xi, 并转基因它与 > 6万 shRNAs 目标 > 2.5万基因的图书馆。我们发现30基因的潮霉素在 B 选择下赋予生存优势, 表明它们在控制MeCP2 和 X 染色体沉默中的作用。这些结果通过传感报告的细胞线与单独发夹和评估激活的无声荧光素酶记者证实。
由于XIST?...

女性遗传性精神损害最常见的形式之一, Rett 综合症, 是由MeCP2中的异型突变引起的, X 染色体基因编码了正常神经元功能所必需的蛋白质1。治疗这种紊乱的一种潜在的方法是重新激活在 Xi 上的野生型MeCP2等位基因, 因为MeCP2表达式的恢复被证明可以逆转这种疾病的老鼠模型的神经缺陷1,2,4. 然而, 在生物体34的整个生命周期中, 女性细胞中的两个 X 染色体之一的表观遗传沉默是严格维持的, 而一个 Xi 基因的健壮重新激活可能需要一个主要多种表观遗传调控途径的中断。
为了确定维护MeCP2沉默所需的因素, 我们首先开发了一种转基因小鼠模型, 它携带MeCP2-荧光素酶-潮霉素抗性基因融合(MeCP2-LUC-HR) 对两个 x 染色体之一 (xMeCP2-LUC-HR/XMeCP2)5。虽然从融合结构中表达的MeCP2被证明是不稳定的, 并导致函数丢失, 但表型在 hemizygous 雄性 (X MeCP2-LUC-HR/Y) 中模仿 MeCP2 删除, 报告基因的表达 在与内源 MeCP25的表达式一致的模式下易于检测。然后, 我们在非活动 X 染色体上生成永生化成纤维细胞克隆, 并证实前者表达的野型 MeCP2 而非记者构造;后者的逆是真的。当暴露在已知的 DNA 去甲基化剂被称为废除基因沉默, 5-azacytidine (5-AZA), 细胞与记者在 Xi ("记者细胞") 获得活性的生物发光检测, 表明我们的构造可以重新激活, 因此用于基因筛选。
接下来, 我们为MeCP2沉默的监管者开发了一个高通量的遗传屏幕。记者细胞线首先感染了一个包含 > 6万不同 shRNAs 靶向 > 2.5万基因的逆转录病毒库, 整个鼠标基因组5,6, 然后受到潮霉素 B 选择。用下一代测序方法比较了前、后选择样品中的发夹频率, 因为在潮霉素 B 选择的情况下, 记者重新激活赋予了增长优势, 导致了责任发夹的丰富。利用这一方法, 我们确定了30个与MeCP2沉默有关的基因, 随后通过传感与单个发夹的报告细胞并测量其荧光素酶活性来证实这一发现。

<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&#039;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&amp;ndash;401&amp;ndash;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

通过插入到染色质中的报告基因的正向基因筛已被广泛用于研究模型生物体的表观遗传控制机制。技术, 包括短发夹 rna (shRNAs) 和聚集定期 interspaced 短回文重复 (CRISPR) 已启用这样的屏幕在双倍哺乳动物细胞。在这里, 我们描述了一个大规模的 shRNA 屏幕, 用于调节 X 染色体失活 (XCI), 使用小鼠细胞系与萤火虫荧光素酶和潮霉素抗性基因敲在 C 终点的甲基 CpG 结合蛋白 2(MeCP2) 基因不活跃的 X 染色体 (Xi)。在潮霉素 B 选择的情况下, 重新激活该构造, 使我们能够屏蔽一个大型 shRNA 库, 并通过测量其选择后的丰富度来确定重新激活记者的发夹。下一代测序。丰富的发夹, 然后通过测试他们的能力激活的荧光素酶记者在西安单独验证。

小鼠肌腱成纤维细胞是从先前描述的 xMeCP2-LUC-HR/XMeCP2雌性小鼠身上收获的, 它由 HPV-16 virus7 的逆转录病毒传导 E6 和 E7 癌基因所永生化, 并通过限制性稀释进行克隆, 并测试荧光素酶活性和野生型 MeCP2 蛋白的表达 (图 1A 和 1B)。荧光素酶活性是健壮的, 如果记者是在 Xa, 并不能察觉, 如果在西安;本机 MeCP2 表达了一个相互的模式。我们选择了一个克隆?...

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

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

我们报告一个小发夹 RNA (shRNA) 和下一代测序协议, 以确定在小鼠细胞系的 X 染色体失活的监管者与萤火虫荧光素酶和潮霉素抵抗基因融合到甲基 CpG 结合蛋白 2 (MeCP2)非活性 X 染色体上的基因。

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

pooled-shrna-screen-for-reactivation-mecp2-on-inactive-x

Research

JoVE Journal

Genetics

7.2K 次观看.  Fred Hutchinson Cancer Research Center. 我们报告一个小发夹 RNA (shRNA) 和下一代测序协议, 以确定在小鼠细胞系的 X 染色体失活的监管者与萤火虫荧光素酶和潮霉素抵抗基因融合到甲基 CpG 结合蛋白 2 (MeCP2)非活性 X 染色体上的基因。

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

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

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 females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

非随机小鼠模型在非活性 X 染色体上对 Mcp2 的药理再激活

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 ...

科研

遗传学

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

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 cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳动物细胞中基于CRISPR的聚集基因屏幕

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 ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

癌症研究

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

MeCP2蛋白质变异的电化学发光测定

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

生物化学

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

基因组规模筛选，用于识别和比较肿瘤中的治疗靶点

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

作者聚焦:通过基因组规模 shRNA 筛选寻找恶性周围神经鞘瘤的新治疗靶点 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

生物学

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

使用工程 HEK 细胞筛选激活 MRGPRX2 的肽

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

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非随机小鼠模型在非活性 X 染色体上对 Mcp2 的药理再激活

哺乳动物细胞中基于CRISPR的聚集基因屏幕

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

MeCP2蛋白质变异的电化学发光测定

合并的shRNA文库筛选，以确定调节耐药性表型的因素

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

使用工程 HEK 细胞筛选激活 MRGPRX2 的肽