这项工作得到了国立卫生研究院/国家神经疾病 & 中风研究所 (NIH/一) 的资助, R37-NS094804、R01-NS074387、R21-NS091555 M.G.C.;NIH/一授予 R01-NS076991、R01-NS082311 和 R01-NS096756 P.R.L.;NIH/一 R01-EB022563;神经外科部;利亚快乐的心, 和乍得虽然基金会 M.G.C. 和 P.R.L. RNA 生物医学补助金 F046166 M.G.C. F.M.M. 是支持 F31 NIH/NINDS-F31NS103500。R.I.Z.-五。由 NIH/研究院资助5T34GM007821-37。

这里描述的所有方法都得到了密歇根大学机构动物保育和使用委员会 (IACUC) 的批准。
1. 产生脑肿瘤 NS 和培养条件。
<ol><li>准备神经干细胞培养基 (国安局) 与 Dulbecco 的改良鹰 Medium/F12B27 补充剂 (1x), N2 补充剂 (1x), 和抗菌试剂。在与人类重组内皮生长因子 (EGF) 和碱性成纤维细胞生长因子 (增殖素) 的使用日, 在最终浓度为 20 ng/毫升时, 补充国安局培养基。</li>
	<li>利用睡眠美容转系统诱导小鼠脑肿瘤, 将基因或 shRNA 靶向肿瘤抑制物的质粒编码, 注入新生儿的侧脑室, 产生自发性脑肿瘤9。选择一个大肿瘤的老鼠, 这是证实与生物发光成像。 注: 有关睡眠美容转系统和质粒结构的详细信息, 可在 Calinescu et al.中找到。9。<ol>
<li>为图像的小鼠, 注入100µL 30 毫克/毫升素溶液腹腔使用26口径针。</li>
		<li>5分钟后注射素, 麻醉的动物使用麻醉室氧/异氟醚流量设置为2% 异氟醚。</li>
		<li>当动物被麻醉 (通常2-3 分钟), 安置动物在生物发光房间与氧异氟醚设置到2% 异氟醚。如果这些动物的麻醉时间超过5分钟, 请使用眼部药膏来预防麻醉时的干燥。</li>
		<li>使用活体光学成像系统对小鼠进行图像处理, 其参数如下: 自动曝光、大分和孔径 f=1。考虑它的参量大肿瘤是生物发光 > 106光子/s/cm2/sr。</li>
	</ol>
</li>
	<li>用过量的异氟醚吸入麻醉剂在密闭的室内安乐, 用脚趾夹紧检查动物的麻醉, 并斩首鼠标。</li>
	<li>提取大脑和解剖它如前面所描述的 Calinescu et al.9。</li>
	<li>将大脑放置在10厘米的培养皿中。如果肿瘤诱导表达荧光蛋白, 使用荧光解剖显微镜设置适当的荧光通道和0.3X 放大倍数, 否则识别肿瘤肿块的不同组织的颜色和质地。用手术刀和镊子将肿瘤组织从正常的大脑中分离出来。</li>
	<li>将肿瘤切成小块在培养皿中。将解剖肿瘤放置在1.5 毫升的管中, 300 µL。</li>
	<li>使用一次性塑料颗粒杵, 适合于壁锥形离心管轻轻质肿瘤。加入1毫升的细胞剥离培养基, 并孵育样品5分钟在37° c。 注意: 使用杵可能会杀死一些细胞。如果读者想最大化细胞的生存能力, 使用杵可以省略。</li>
	<li>通过70µm 细胞过滤器通过1.7 步的细胞悬架, 并用25毫升的国安培养基清洗滤网。</li>
	<li>离心悬浮在 300 x g 在室温下5分钟, 醒酒上清和再悬浮颗粒成6毫升的国安会培养基。</li>
	<li>板肿瘤细胞悬浮从步1.9 入 T-25 文化烧瓶和文化它在37° c 在一个组织培养孵化器以95% 空气大气和 5% CO2。通常在3天之后, 会有一些死细胞、黏附的细胞和一些形成 NS 的细胞混合物在培养基中漂浮。</li>
	<li>将细胞悬浮液转移到15毫升的锥形管中, 只收集到底部的细胞, 使用微, 再将其重新印在 T-75 的组织培养瓶上。</li>
	<li>保持相对高密度的单元格 (T-25 中的 1-3 X 106单元格)。通过细胞时, 他们是汇合。 注: 70-100 是由球体大小决定的。大球体的黑色中心意味着中心有死细胞, 并表明细胞应立即传代。</li>
	<li>每三天增加生长因子 (EGF 和碱性-20/毫升)。当细胞不汇合, 培养基变浅橙色时, 改变培养基。</li>
	<li>为了改变培养基, 收集旧介质, 在室温下离心 300 x g, 在5分钟内将细胞颗粒重新悬浮, 并在1.1 步中指定的浓度中补充 EGF 和生长因子。 注: 如果球体在中至高 70-100, 但尚未准备好传代 (球体尺寸较小, 直径 < 100 µm), 则小球可分为两个烧瓶。</li>
	<li>通过细胞, 离心细胞在 300 x g 为5分钟在室温下, 醒酒上清液和孵育细胞脱离介质5分钟在37° c。<ol>
<li>加入5毫升的平衡盐溶液稀释剥离介质, 并离心 300 x g, 在室温下5分钟。</li>
		<li>醒酒上清液, 在18毫升的重中, 将细胞颗粒分成三 T-25 烧瓶。</li>
	</ol>
</li>
	<li>要冻结细胞的等分, 将细胞与步骤1.15 分离, 并在1毫升等分中冷冻90% 胎牛血清和10% 二甲基亚砜的细胞。将细胞放在冰上10分钟, 然后保持细胞-20 ° c 30 分钟,-80 ° c 为1天, 慢慢冷却细胞。第二天, 将细胞转移到液态氮储存容器。</li>
</ol>2. 原片
<ol><li>染色质制剂<ol>
<li>在 T-75 的培养基中, 在37° c 的15毫升的组织培养箱中, 在95% 空气和 5% CO2的环境下, 在 ns 成为汇合体后, 将其培养成 ns。一个汇合的 T-75 板将产生 3-5 X 106单元。</li>
		<li>当细胞成为汇合, 离心机 NS 在 300 x g 为5分钟, 醒酒上清和增加1毫升细胞离解介质。在37° c 孵育5分钟的细胞, 添加5毫升培养基, 并使用例10对单元格进行计数。 注意: 每个沉淀 (IP) 反应需要 1 x 106单元格。请注意, 前免疫血清或 IgG 控制也必须包括11。</li>
		<li>离心的 ns 在一个1.5 毫升离心管在 300 x g 5 分钟, 醒酒上清和再悬浮 ns 颗粒与1毫升的平衡盐溶液和转移悬浮到低蛋白结合1.5 毫升离心管。</li>
		<li>离心机 NS 在 300 x g 为5分钟, 醒酒上清和重新悬浮在95µL 消化缓冲液中的细胞颗粒 (50 毫米三盐酸, pH 8.0, 1 毫米 CaCl2, 0.2% 聚乙二醇苯基醚) 每 1 x 106细胞补充的蛋白酶高浓度抑制剂鸡尾酒 (1:100)。立即吸管细胞, 防止结块, 避免产生气泡。</li>
		<li>重 MNase 50% 甘油, 使1毫克/毫升溶液 (1.15 单位/µL, 储存在-20 ° c), 将被称为 "1x" 股票。做一个 "0.1x" 的股票, 做第二1:9 稀释与50% 甘油 (e. g, 900 µL 的 "1x" MNase 和100µL 50% 甘油), 并将其存储在-20 ° c。</li>
		<li>混合5µL 的 "0.1x" MNase 和145µL 的消化缓冲液。保持酶在冰上的所有时间。在消化缓冲液中加入5µL 稀释 MNase 每 1 x 106单元格。弹管混合和放置在37° c 块的管正好12分钟. 一次处理样品, 确保准确的孵化时间为12分钟。</li>
		<li>每 1 x 106单元格添加10µL 10x MNase 停止缓冲器 (110 毫米三盐酸, pH 8.0, 55 毫米 EDTA)。从这一点开始, 样品必须放在冰上。</li>
		<li>添加110µL "2x 帕缓冲器" (280 毫米氯化钠, 1.8% 聚乙二醇苯基醚, 0.2% 十二烷基硫酸钠 (SDS), 0.2% Na-胆酸, 5 毫米乙二醇二 (β-氨基乙基醚)-n, n, n, n, 乙酸酸 (EGTA); 储存在4° c) 补充蛋白酶抑制剂鸡尾酒在高浓度 (1:100) 每 1 x 106细胞。弹管混合。</li>
		<li>离心管15分钟在一个离心在4° c 和 1700 x g. 将上清液转移到新的常规1.5 毫升离心管 (低蛋白结合离心管不再需要) 并将它们储存在冰上。丢弃颗粒。</li>
	</ol></li>
	<li>沉淀 (IP)<ol>
<li>混合蛋白 a 和蛋白 G 磁性珠子在1:1 的比例。每个 IP, 准备25µL 的珠子。</li>
		<li>通过加入1毫升的帕缓冲液 (10 mm pH 8.0, 1 毫米 EDTA, 140 毫米氯化钠, 1% 聚乙二醇苯基醚, 0.1% SDS, 0.1% Na 胆酸; 储存在4° c), 辅以低浓度的蛋白酶抑制剂 (1:1000)。</li>
		<li>使用磁铁允许珠子从洗涤液中分离, 醒酒洗涤液 (约1分钟)。执行清洗步骤两次。</li>
		<li>使用帕缓冲剂补充蛋白酶抑制剂 (1:1000) 将珠子重新悬挂到原来的体积。</li>
		<li>如有必要, 用蛋白酶抑制剂 (1:1000) 补充帕缓冲液稀释染色质, 使每个 ip 都有100-200 µL 的体积. 保留10% 的体积使用每 ip 染色质为输入。 注: 例如, 如果每 IP 使用200µL, 则应为输入保留20µL 的稀释染色质。共四 IPs, 总染色质体积应稀释到820µL。</li>
		<li>准备输入。添加100µL 的 TE 缓冲器 (10 毫米三盐酸 pH 8.0, 1 毫米 EDTA) 补充蛋白酶 K (0.5 毫克/毫升) 到输入和孵化输入在55° c 为 1 h。 注意: 步骤 2.2. 6-2. 2.7 可以与步骤2.3.4 和2.4.1 一起执行。</li>
		<li>按照制造商的说明和洗在试剂盒中提供的50µL 洗脱缓冲液, 使用 PCR 纯化试剂盒净化输入。</li>
		<li>使用 microvolume 分光光度计测量输入的浓度, 并将结果记录在笔记本中。空白与洗脱缓冲从成套工具。 注意: 在鼠标球, 我们使用了大约6µg 的 DNA 为每个 IP。</li>
		<li>运行在1% 凝胶或负载1µL 在 DNA Bioanalyzer 使用标准设置的输入。将余数存储为在下游应用程序中用作输入。</li>
		<li>添加10µL 的洗涤蛋白 A & G 磁性珠子为每个 ip 和孵化 ip 为1小时在4° c。 注: 例如, 为三 IPs 添加30µL 的蛋白质 A & G 磁性珠子。</li>
		<li>把样品放在磁铁上, 让珠子分开。通过将先前确定的量转移到新的1.5 毫升管中来分离染色质。</li>
		<li>加入抗体, 用塑料石蜡膜包裹瓶盖, 避免蒸发。在4° c 的夜间孵育样品, 旋转 20 rpm。 注: 浓度应根据制造商的推荐经验来确定。</li>
		<li>第二天, 在室温下使用迷你离心机旋转管子, 2000 x g, 十年代。然后加入10µL 的珠子到每个 ip 和孵化 ip 为3小时在4° c 与自转在20转每分钟。</li>
	</ol></li>
	<li>IP 洗涤和蛋白质消化<ol>
<li>添加150µL 的帕缓冲剂补充与蛋白酶抑制剂在低浓度 (1:1000) 到每个 ip 和孵化 ip 在4° c 与自转在20转每分钟为 5 min. 将样品放在磁性的支架上, 让磁珠分开。用吸管去除上清液。重复这 tep 五洗。</li>
		<li>添加150µL 氯化缓冲器 (250 毫米氯化, 10 毫米三, pH 8.0, 1 毫米 EDTA, 0.5% NP-40, 0.5% Na 胆酸; 贮存在4° c) 在低浓度 (1:1000) 的补充与蛋白酶抑制剂, 在4° c 的每 ip 和孵育 ip 以自转在 20 rpm 为 5 min. 安置样品在磁性的立场和允许磁珠分开。用吸管去除上清液。</li>
		<li>添加150µL 的冷 TE (无蛋白酶抑制剂) 和孵化的样品在4° c 旋转在 20 rpm 为5分钟. 将样品放在磁性支架上, 并允许磁珠分开。用吸管去除上清液。</li>
		<li>在最后的洗涤步骤, 重新暂停样品在100µL 的 TE 缓冲补充蛋白酶 K (0.5 毫克/毫升) 和孵化样品在55° c 为1小时。</li>
	</ol></li>
	<li>DNA 纯化与芯片定量实时 PCR (qPCR)<ol>
<li>利用 PCR 纯化试剂盒净化 immunoprecipitated DNA。在从试剂盒中添加 DNA 结合缓冲器到每个样品后, 将管放在磁铁上, 等待磁珠聚合, 然后将上清液转移到清理柱上。按照制造商的说明和洗在50µL 的洗脱缓冲器提供的套件。</li>
		<li>要检查 IP 是否成功, 请执行 qPCR。理想情况下, 底漆应设计为正负控制区域。 注: 例如, 对于使用 H3K4me3 和 H3K27me3 执行的 IP, 以下示例应在 qPCR 上运行: H3K4me3、H3K27me3 和 IgG 芯片 dna、输入 DNA 和无模板控制 (NTC), 使用引物来丰富区域与 H3K4me3 (阳性控制) 和用 H3K4me3 (负控制) 丰富区域;同样, 对于 H3K27me3。</li>
		<li>按照标准协议执行 qPCR12。简要地, 执行 qPCR 使用 SYBR 绿色大师混合, 0.5 µL 10 µM 工作集中每向前和反向底漆和2µL 芯片或输入脱氧核糖核酸。使用实时 PCR 系统读取车牌。在表 2中提供了入门序列。Gapdh 引物序列从黄et al。使用了13。</li>
		<li>分析与输入相关的芯片数据,即百分比输入方法 (% IP)14。使用以下公式计算百分比输入: 调整的输入 = 平均 ct (输入)-日志2(DF) DF 是起始输入的稀释因子, DF = 总的 IP 容量/输入量 % ip = 100×2 ^ (被调整的输入-Ct (IP)) 其中 Ct 是阈值因子。</li>
	</ol></li>
</ol>

<ol>
	<li>Schwartzentruber, 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=Driver+mutations+in+histone+H3.3+and+chromatin+remodelling+genes+in+paediatric+glioblastoma.">Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.</a> Nature. 482 (7384), 226-231 (2012).</li><li>Bjerke, L., 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=Histone+H3.3.+mutations+drive+pediatric+glioblastoma+through+upregulation+of+MYCN.">Histone H3.3. mutations drive pediatric glioblastoma through upregulation of MYCN.</a> Cancer Discov. 3 (5), 512-519 (2013).</li><li>Bender, 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=Reduced+H3K27me3+and+DNA+hypomethylation+are+major+drivers+of+gene+expression+in+K27M+mutant+pediatric+high-grade+gliomas.">Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas.</a> Cancer Cell. 24 (5), 660-672 (2013).</li><li>Maleszewska, M., Kaminska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+glioblastoma+an+epigenetic+malignancy?.">Is glioblastoma an epigenetic malignancy?.</a> Cancers (Basel). 5 (3), 1120-1139 (2013).</li><li>Gilfillan, G. D., 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=Limitations+and+possibilities+of+low+cell+number+ChIP-seq.">Limitations and possibilities of low cell number ChIP-seq.</a> BMC Genomics. 13, 645 (2012).</li><li>Thorne, A. W., Myers, F. A., Hebbes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+chromatin+immunoprecipitation.">Native chromatin immunoprecipitation.</a> Methods Mol Biol. 287, 21-44 (2004).</li><li>Turner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mapping Protein/DNA Interactions by Cross-Linking. , (2001).</li><li>Tseng, Z., Wu, T., Liu, Y., Zhong, M., Xiao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+native+chromatin+immunoprecipitation+to+interrogate+histone+variant+protein+deposition+in+embryonic+stem+cells.">Using native chromatin immunoprecipitation to interrogate histone variant protein deposition in embryonic stem cells.</a> Methods Mol Biol. 1176, 11-22 (2014).</li><li>Calinescu, A. 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=Transposon+mediumted+integration+of+plasmid+DNA+into+the+subventricular+zone+of+neonatal+mice+to+generate+novel+models+of+glioblastoma.">Transposon mediumted integration of plasmid DNA into the subventricular zone of neonatal mice to generate novel models of glioblastoma.</a> J Vis Exp. (96), (2015).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cell+number+by+hemocytometry+counting.">Estimation of cell number by hemocytometry counting.</a> CSH Protoc. 2006 (1), (2006).</li><li>Landt, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22 (9), 1813-1831 (2012).</li><li>Kubista, M., 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+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol Aspects Med. 27 (2-3), 95-125 (2006).</li><li>Hwang, W. W., 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=Distinct+and+separable+roles+for+EZH2+in+neurogenic+astroglia.">Distinct and separable roles for EZH2 in neurogenic astroglia.</a> Elife. 3, e02439 (2014).</li><li>Haring, M., 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=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.</a> Plant Methods. 3, 11 (2007).</li></ol>

作者没有什么可透露的。

这里提供的协议将使用户能够在来自基因工程脑肿瘤的 NS 上执行本机芯片。与交叉连接芯片相比, 这个协议是有限的研究与 DNA 紧密关联的蛋白质6。所使用的单元格数可以根据需要进行修改, 并且可以扩展协议。我们每 IP 使用 1 X 106单元, 但是, 本机芯片也可以执行, 只要有 4 X 104单元格5。在本协议中, 我们通过 qPCR 分析芯片 DNA;然而, 该协议也可?...

后生事件在胶质瘤中很常见, 可能在肿瘤发病中起重要作用。的确, 在儿科高级别胶质瘤中, 基因组蛋白变体 H3.3 和 H3.1 编码的突变通常发生在1。突变影响组蛋白修饰, 并有主要的后生后果2,3。在青春期到成人频谱, 复发突变的异脱氢酶基因 1/2 (IDH1/2), 一种突变, 抑制α公斤依赖组蛋白和 DNA de methylasaes, 以及基因改变的其他染色质调节器, 如 ATRX 和 DAXX 发生4. 因此, 研究影响后生调节因子的突变是如何改变染色质结构和调节组蛋白修饰的至关重要的, 而这反过来又会对肿瘤细胞的转录产生巨大的影响。
染色质沉淀 (芯片) 是一种功能强大的工具, 用于评估在基因组5,6,7中表观遗传修饰的影响。在原生芯片中, 染色质是用 micrococcal 核酸 (MNase) 消化的, immunoprecipitated 使用对感兴趣的蛋白质提出的抗体, 然后从 immunoprecipitated 染色质复合体6中纯化出 DNA。细胞在过程中没有固定, 因此这种技术只适用于与 DNA6紧密交互的蛋白质的研究。缺乏交叉链接艾滋病抗体特异性, 因为抗体通常是针对不固定的多肽或蛋白质7。此外, 由于没有交叉链接的步骤, 这就减少了固定非特定的瞬时蛋白质-DNA 相互作用的几率, 而不是规范的7,8。芯片可以用来识别在特定的基因组区域中蛋白修饰的丰富性。在这里, 我们详细介绍了一个在球 (NS) 中由基因改造的小鼠胶质瘤模型来执行本机芯片的协议。

<table><tbody><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent</td><td>G2946-90004</td><td>bioanalyzer</td></tr><tr><td>AccuSpin Micro 17R, refrigerated</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>C57BL/6</td><td>Taconic</td><td>B6-f</td><td>C57BL/6 mouse</td></tr><tr><td>Calcium Chloride</td><td>Aldrich</td><td>22350-6</td><td>buffer reagent</td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>Goldbio</td><td>LuckK-1g</td><td></td></tr><tr><td>DiaMag1.5 magnetic rack</td><td>Diagenode</td><td>B04000003</td><td>for magnetic bead washes</td></tr><tr><td>DiaMag Rotator EU</td><td>Diagenode</td><td>B05000001</td><td>rotator</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>11330-057</td><td>NSC component</td></tr><tr><td>Dynabeads Protein A</td><td>Thermo Fisher Scientific</td><td>10001D</td><td>protein A magnetic beads</td></tr><tr><td>Dynabeads Protein G</td><td>Thermo Fisher Scientific</td><td>10003D</td><td>protein G magnetic beads</td></tr><tr><td>EGF</td><td>PeproTech</td><td>AF-100-15</td><td>prepare 20 &amp;mu;g/mL stock in 0.1% BSA and aliquot.</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>E-4884</td><td>buffer reagent</td></tr><tr><td>ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid) (EGTA)</td><td>Sigma</td><td>E-4378</td><td>buffer reagent</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>Applied Biosystems</td><td>4385612</td><td>qPCR reagent</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10437028</td><td>for freezing cells</td></tr><tr><td>FGF</td><td>PeproTech</td><td>100-18b</td><td>Prepare 20 &amp;mu;g/mL stock in 0.1% BSA and aliquot.</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11008-13</td><td>for dissection of tumor</td></tr><tr><td>Glycerol, MB Grade</td><td>EMD- Millipore</td><td>356352</td><td></td></tr><tr><td>H3K4me3</td><td>Abcam</td><td>Ab8580</td><td></td></tr><tr><td>H3K27me3</td><td>Millipore</td><td>07-449</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-103</td><td>balanced salt solution</td></tr><tr><td>Fluriso</td><td>VETone</td><td>501017</td><td>inhalation anesthetic</td></tr><tr><td>Ivis Spectrum</td><td>Perkin-Elmer</td><td>124262</td><td>in vivo optical imaging system</td></tr><tr><td>Hyqtase</td><td>HyClon</td><td>SV3003001</td><td>cell detachment media</td></tr><tr><td>Lithium Chloride</td><td>Sigma</td><td>L8895</td><td>buffer reagent</td></tr><tr><td>Low binding microtubes</td><td>Corning Costar</td><td>CLS3207</td><td>low protein binding microcentrifuge tube</td></tr><tr><td>Microcentrifuge tube</td><td>Fisher</td><td>21-402-903</td><td>regular microcentrifuge tube</td></tr><tr><td>Micrococcal Nuclease</td><td>Thermo Fisher Scientific, Affymetrix</td><td>70196Y</td><td>each batch may differ; purchase sufficient amount for experiments and aliquot.</td></tr><tr><td>N2</td><td>Gibco</td><td>17502-048</td><td>NSC component</td></tr><tr><td>Normal Rabbit IgG</td><td>Millipore</td><td>12-372</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>NOL-36-063</td><td>anti-microbial agent, use at 0.1 mg/mL.</td></tr><tr><td>NP-40 (Igepal CA-630)</td><td>Sigma</td><td>18896-50ML</td><td>buffer reagent</td></tr><tr><td>Kimble Kontes Pellet Pestle</td><td>Fisher Scientific</td><td>K749515-0000</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td>aliquot and store at -20 &amp;deg;C.</td></tr><tr><td>Protinase K</td><td>Sigma-Aldrich</td><td>P2308</td><td>make 10 mg/mL stock in water; aliquot and store at -20 &amp;deg;C.</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>DNA purification kit</td></tr><tr><td>Scalpel</td><td>Fine Science Tools</td><td>10007-16</td><td>for dissection of tumor</td></tr><tr><td>Sodium Chloride</td><td>VWR</td><td>0241-5KG</td><td>buffer reagent</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D670-25G</td><td>buffer reagent</td></tr><tr><td>Sodium Dodecyl sulfate (SDS)</td><td>Sigma</td><td>L-4390</td><td>buffer reagent</td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>Bp152-1</td><td>buffer reagent</td></tr><tr><td>Triton X-100</td><td>Thermo Fisher Scientific</td><td>BP 151-500</td><td>polyethylene glycol octylphenyl ether</td></tr><tr><td>Standard Mini Centrifuge</td><td>Fisherbrand</td><td>12-006-901</td><td>standard mini centrifuge</td></tr><tr><td>SZX16 microscope</td><td>Olympus</td><td>SZX16</td><td>flourescent dissecting microscope</td></tr><tr><td>ViiA 7 Real-Time PCR System with Fast 96-Well Block</td><td>Applied Biosystems</td><td>4453535</td><td></td></tr><tr><td>Nanodrop One</td><td>Thermo-Fisher Scientific</td><td>ND-ONEC-W</td><td></td></tr></tbody></table>

native chromatin immunoprecipitation using murine brain tumor neurospheres

后生修饰可能参与胶质瘤的发展和进展。癌基因和肿瘤抑制因子的促发和调控区的甲基化和再乙酰性的改变, 可能会导致在脑肿瘤的发病机制中的表达改变, 并发挥重要作用。原生染色质沉淀 (芯片) 是一种流行的技术, 允许检测的修改或其他蛋白质紧紧绑定到 DNA。与交联芯片不同的是, 在原生芯片中, 细胞不能用甲醛来将蛋白质与 DNA 进行共价键连接。这是有利的, 因为有时交联可能修复的蛋白质, 只有短暂的互动与 DNA, 并没有功能意义的基因调控。此外, 抗体通常是针对不固定的多肽。因此, 原片中抗体特异性增加。然而, 重要的是要记住, 本机芯片只适用于研究组蛋白或其他与 DNA 紧密结合的蛋白质。本协议描述了鼠脑肿瘤球的原染色质沉淀。

在图 1中, 在脑肿瘤细胞 Katushka 阳性的脑瘤中产生的肿瘤 NS 的图示。图 2是整个芯片技术的示意图表示。图 3显示了来自脑肿瘤 NS 的染色质的代表性结果, MNase 为12分钟, 产生了大多数单、di 和三核。在芯片上, 可以对芯片进行 qPCR, 并输入 DNA 样本。图 4显示了来自 qPCR glyceraldehyde-3-phosp...

Watch this Scientific Journal Video about Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres at JoVE.com

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

利用小鼠脑肿瘤球的原发染色质沉淀

后生机制在胶质瘤中经常被改变。染色质沉淀可用于研究基因改变的后果, 胶质瘤的改变导致的组蛋白修饰, 调节染色质结构和基因转录。本协议描述了小鼠脑肿瘤球的原发染色质沉淀。

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

native-chromatin-immunoprecipitation-using-murine-brain-tumor

Research

JoVE Journal

Cancer Research

7.6K 次观看.  University of Michigan Medical School. 后生机制在胶质瘤中经常被改变。染色质沉淀可用于研究基因改变的后果, 胶质瘤的改变导致的组蛋白修饰, 调节染色质结构和基因转录。本协议描述了小鼠脑肿瘤球的原发染色质沉淀。

视频: Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

科研

癌症研究

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

从人死后脑组织神经元细胞核分离

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

生物学

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

神经科学

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

医学

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of neuro-oncology. One of the most widely used techniques is the orthotopic intracranial injection, a method that allows investigating of tumor growth, invasion, and dissemination within a controlled setting. This technique involves transplanting tumor cells from a specific patient region into the corresponding anatomical site in an animal. By doing so, these orthotopic brain tumor models offer a unique advantage, as they more accurately replicate cancer's biological behavior and its interactions with the brain environment seen in human patients. This makes them especially valuable for preclinical therapeutic testing, where a close resemblance to the clinical scenario is essential for evaluating potential treatments. This protocol shares experiences in developing patient-derived xenograft (PDX) models for pediatric brain tumors, including diffuse midline glioma (DMG), glioblastoma (GBM), medulloblastoma, and ependymoma. This method delineates the procedure for conducting intracranial stereotaxic injections in mice, ensuring the correct targeting of the injection site within the brain. Additionally, we describe the post-procedural monitoring system employed to detect signs of successful tumor engraftment. Following tumor injection, a rigorous monitoring system is implemented to observe the animals for any signs of neurological impairment, behavioral changes, and/or weight loss, which are common indicators of tumor progression. This system allows for timely intervention and provides critical data regarding the tumor's growth dynamics. By refining these models and protocols, we aim to enhance the reliability and translational potential of preclinical studies, contributing to the development of more effective treatments for pediatric CNS tumors.

The development of orthotopic pediatric brain tumor models requires meticulous precision, using a stereotaxic device to implant cancer cells precisely. The methodology presented here outlines the steps involved in preparing brain tumor cells, performing intracranial injections, and implementing a post-operative monitoring system to assess brain tumor engraftment.

The development of clinically relevant and reliable models for central nervous system (CNS) tumors has been pivotal in advancing the field of ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

免疫与感染

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

<a href='https://app.jove.com/v/58931/evaluation-biomarkers-glioma-immunohistochemistry-on-paraffin'>Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures.&#160;J. Vis. Exp. (2019).</a>This video demonstrates an immunohistochemical technique for labeling tumor biomarkers in paraffin-embedded brain tumor neurospheres. The neurosphere sections undergo paraffin removal and rehydration, followed by treatment to expose biomarker sites. Primary and secondary antibodies are then applied to label these biomarkers. The secondary antibodies are bound by avidin-conjugated peroxidase enzymes, which oxidize chromogenic substrates to indicate biomarker locations. Finally, nuclear counterstaining and slide preparation are completed for microscopic analysis.

Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

Native Chromatin Immunoprecipitation of Neurosphere Cells

<a href='https://app.jove.com/v/57016/native-chromatin-immunoprecipitation-using-murine-brain-tumor'>Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).</a>This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).This video ...