我们感谢亨利埃特 Bertemes 在实验室中帮助建立 CGN 种植协议。本方法通过对电视的资助, 由 DFG 资助的 CRC992 医学遗传学研究支持。作者承认弗莱堡星系小组的支持: Pavankumar Videm, bj Grüning 和 Prof., 生物信息学, 德国弗赖堡大学, 由合作研究中心资助的992医学遗传学 (Backofen 补助金 DFG 992/1 2012)并且德国联邦教育和研究部 (BMBF 授予 031 A538A RBC (de。NBI))。

弗莱堡大学的动物福利委员会和地方当局批准了以下协议中提到的所有动物实验 (G12/13, G16/11)。
1. 准备工作
<ol><li>
党分离制剂
	<ol>
<li>建立定时交配, 以获得胚胎在不同阶段的皮层发育 (E11.5 和 E14.5)。使用小鼠的应变 NMRI (海军医学研究所), 这是至少8周的老。交配后, 考虑一个积极的阴道堵塞在 E0.5。</li>
		<li>要有足够的材料, 在 E12.5 或 E14.5 的皮质上用一小块 NMRI 鼠 H3K79me2 芯片。对于以后的胚胎阶段, 用一小块做更多的芯片分析 (平均 NMRI:10 胚胎)。</li>
		<li>冷 Hank´s 平衡盐溶液 (HBSS) 和磷酸盐缓冲盐水 (PBS) 到4° c (长期存贮在 RT)。</li>
		<li>平衡4° c 储存的胰蛋白酶-EDTA (0.05% w/v) 到37° c。</li>
		<li>
准备皮质细胞培养基 (CCM):补充培养基的神经元 (见材料表) 的最终浓度为 2% (v/v) B-27 补充, 5 µg/毫升的脂蛋白-转铁蛋白, 1 µg/毫升谷胱甘肽, 0.5 毫米 l-谷氨酰胺, 0.8 µg/毫升超氧化物歧化酶, 1% (v/v)青霉素-链霉素-霉素抗生素混合物。储存在4° c。为实验, 平衡介质到37° c。</li>
		<li>解冻胎儿小牛血清 (FCS), 分它 (50 毫升每) 和平衡一分到37° c (长期存贮在-20 ° c)。</li>
		<li>在 H2O 中为单元格区域性准备 dnasei 1 (10 毫克/毫升) 的库存。商店等分在-20 ° c。</li>
		<li>如果需要, 溶解特定的 DOT1L 抑制剂 EPZ-5676 或 SGC0946 在亚甲基亚砜的股票浓度为100毫米。将1-5 µM 的末端浓度应用于0天的细胞, 每两天重新涂抹一次抑制剂。</li>
		<li>为 CPC 培养, 涂层细胞培养板材 (6 井或12井) 与聚 l-鸟氨酸氢溴酸盐 (1 毫克/毫升在150毫米硼酸酸碱度 8.4) 为至少 1 h 在 RT 和之后与层粘连蛋白 (1 毫克/毫升) 在 CCM 媒介在37° c 隔夜。</li>
	</ol>
</li>
	<li>
CGNP 分离制剂
	<ol>
<li>组织适当的小鼠交配, 以产生 P5-P7 的动物来隔离小脑 (每个芯片和条件3-5 动物)。</li>
		<li>在 HBSS 缓冲液中加入6毫克/毫升葡萄糖, 准备 HBSS/葡萄糖。</li>
		<li>制备 CGNP 细胞培养基 (CGM), 加入 1% (v/v) N2 补充剂, 1% (v/v) 青霉素-链霉素-霉素, 25 毫米氯化钾和 10% FCS 到 DMEM-F12。为 CGNP 耕种准备 CGM 媒介, 不用 FCS, 但包括0.6 µg/毫升声波刺猬 (嘘) (cgm-嘘) 和平衡它到37° c, 当需要时。</li>
		<li>6层细胞培养板, 100 µg/毫升的聚-d-赖氨酸1-2 小时用于胶质细胞去除。事后冲洗两次, 无菌 ddH2O, 让盘子晾干。在4° c 的最大一周贮存板材。</li>
		<li>为培养 CGNPs 外套 6-好细胞培养板材与聚 l-鸟氨酸 (0.1 毫克/毫升) 在4° c 隔夜。之后洗涤两次与 H2O 为细胞文化并且让板材烘干。 注: 板材可在4° c 下贮存最多一周。</li>
		<li>准备0.025% 胰蛋白酶 (w/v) 在 HBSS/葡萄糖和平衡它到37° c, 当需要时。</li>
	</ol>
</li>
	<li>
H3K79me2 片的制备
	<ol>
<li>在交联染色质前, 准备好粉煤灰 (PBS 1%, pH 8)。在微波中制备50毫升 PBS, 最大65° c。在不断搅拌期间添加0.5 毫克粉煤灰到 PBS。然后加入50µL 10 M 氢氧化钠, 等到粉煤灰溶解。之后, 添加42.5 µL HCl (37% v/v) 以获得 pH 值8。再次验证 pH 值, 然后让1% 的粉煤灰溶液到达 RT。</li>
		<li>在无菌 ddH 中分别制备核糖核酸 (1 毫克/毫升) 和蛋白酶 K (20 µg/µL), 在-20 ° c 下储存等分.</li>
		<li>准备以下缓冲器和试剂: PBS 含有0.02% 吐温, 裂解缓冲, 甘氨酸, 稀释缓冲, 芯片缓冲器 1, 芯片缓冲器 2, 芯片缓冲器 3, 和 TE 缓冲器, 并存储在4° c。每次准备洗脱缓冲器。<ol>
<li>准备包含0.02% 补间的 PBS。最多一周在4° c 存储。</li>
			<li>在 pH 8.0、10毫米 EDTA、1% (w/v) 十二烷基硫酸钠 (SDS) 和1x 蛋白酶抑制剂上制备裂解缓冲液。</li>
			<li>准备2.5 米甘氨酸。</li>
			<li>用20毫米的 pH 值 8.0, 150 毫米氯化钠, 2 毫米 EDTA, 1% (v/v) 海卫 X-100, 0.25% (w/v) SDS, 和1x 蛋白酶抑制剂准备稀释缓冲液。</li>
			<li>在 pH 8.0、150毫米氯化钠、2毫米 EDTA、1% (v/v) 海卫 X-100 和 0.2% (w/v) SDS 上使用 20 mm 的三毫米制备芯片缓冲器1。</li>
			<li>准备芯片缓冲器2使用20毫米的 pH 8.0, 500 毫米氯化钠, 2 毫米 EDTA, 1% (v/v) 海卫 X-100 和 0.2% (w/v) SDS。</li>
			<li>准备芯片缓冲器3使用20毫米的 pH 8.0, 250 毫米氯化, 2 毫米 EDTA, 1% (v/v) NP-40 和 1% (w/v) SDS。</li>
			<li>使用20毫米的三 pH 8.0 和2毫米 EDTA 制备 TE 缓冲器。</li>
			<li>准备新的洗脱缓冲液, 含 1% (w/v) SDS, 100 mM NaHCO3。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 神经祖脑组织的分离
<ol><li>
党的分离与可选栽培
	<ol>
<li>通过宫颈脱位安乐妊娠动物, 将胚胎移植到冰冷的 HBSS。</li>
		<li>用剪刀将颅骨取出, 然后将大脑隔离, 然后用小镊子将所有其他脑部部分移除, 如果适用, 用小镊子将皮质 (整个、DT 或 VT) 移除, 如果必要的小剪刀在4° c 的15毫升管中, 将孤立的皮层储存在5毫升的 HBSS 缓冲液中。</li>
		<li>离心分离的脑材料为5分钟在 1000 x g 和4° c。</li>
		<li>用5毫升冰冷的 HBSS 和质用1毫升的吸管尖移向上和向下清洗皮质。如有必要, 切下吸管尖端。</li>
		<li>离心均匀的样品为5分钟在 1000 x g 和4° c。删除 HBSS。</li>
		<li>添加3毫升胰蛋白酶和孵化样品5分钟在37° c。</li>
		<li>添加1毫升 FCS, 5 毫升 CCM, 30 µL dnasei 1 的样品和质的样品与5毫升玻璃吸管移小心上下。</li>
		<li>离心分离的细胞5分钟在 1000 x g 和4° c。丢弃上清, 添加5毫升 CCM, 并再次质样品。</li>
		<li>稀释样品的分, 并与 Neubauer 计数室计数。每个胚胎的平均数量为 4.5 x 106细胞。为被隔绝的党的耕种继续步2.1.10。对于芯片, 立即进行步骤3。</li>
		<li>为耕种, 党在聚 l-鸟氨酸 (0.1 毫克/毫升) 和层粘连蛋白 (1 毫克/毫升) 涂覆的盘子在密度在 8 x 104和 2.5 x 105细胞/cm2在 CCM 培养基和孵育他们在37° c, 5% CO2, 和100% 相对湿度。</li>
		<li>由于党开始分化成神经细胞在细胞培养 (在大约4天以后), 考虑解剖日期作为天在体外0 (天 0)。为了更长的耕种期限, 每第四天改变一半媒介以新鲜的 CCM 媒介。</li>
		<li>应用1-5 µM SGC0946 或 EPZ5676 溶解在二甲基亚砜在0天 (4-5 小时后细胞隔离) 和刷新它每第二天。使用亚甲基亚砜作为对照治疗。如果需要, 通过标准免疫方法测试抑制剂处理的效率。</li>
	</ol>
</li>
	<li>
CGNPs 的分离与可选栽培
	<ol>
<li>用剪刀安乐 P5-7 动物。取出头皮皮肤, 打开头骨, 用小剪刀和镊子把大脑移走。分离小脑并将其转移到冰冷的 HBSS/葡萄糖。取出所有的脑膜和血管, 并将小脑转化为15毫升的 HBSS/葡萄糖的管子。</li>
		<li>洗涤小脑三次与10毫升冰冷 HBSS/葡萄糖 (收集他们的离心在 650 x g 为5分钟, 在4° c) 和质小脑随后轻轻移向上和向下与1毫升吸管 (最大2-3 倍) 得到 0.5-1 mm3碎片.</li>
		<li>在 HBSS/葡萄糖中加入5毫升的0.025% 胰蛋白酶, 并在37° c 水浴下恒温孵育15分钟. 通过加入5毫升 CGM 来停止消化, 并在650° c 以下5分钟的离心法中收集组织。</li>
		<li>去除上清和研制的组织与1毫升吸管尖端在1毫升 CGM 和转移到一个新的15毫升管。 注意: 避免气泡是很重要的。<ol>
<li>加入5毫升 CGM 和孵化混合物2分钟的冰, 以解决组织残余物。将上清液转化为新的15毫升管。添加2毫升 CGM 到剩余的组织和重复研磨程序。</li>
		</ol>
</li>
		<li>池清 (没有组织残余, 大约10毫升) 和离心机在 650 x g 5 分钟在4° c 收集小脑细胞。重10毫升 CGM 中的颗粒。</li>
		<li>由于星形胶质细胞坚持更快, 更强的多聚-d-赖氨酸比 CGNPs, 板在100µg/毫升的聚-d-赖氨酸涂层 6-井板 (高达4毫升每井) 和孵化20分钟在37° c, 以消除星形胶质。</li>
		<li>摇动盘子, 收集上清液。然后离心在 650 x g 为5分钟在4° c 在15毫升管。重10毫升 CGM 中的颗粒, 计数的细胞与 Neubauer 计数室。 注: CGNPs 是圆形, 小, 并显示一个光晕时, 使用相衬显微镜成像。</li>
		<li>如果需要, 种子的细胞在37° c 前预热 CGM 上的聚 l-鸟氨酸涂层板 (3 x 106细胞每 6-井) 和孵化他们在37° c, 5% CO2和100% 相对湿度。 注意: 如果未执行播种, 请继续步骤3.1。<ol>
<li>6-12 小时后, 将介质转换为 CGM-嘘。治疗隔离 CGNPs 6 小时 (或当改变为 CGM 嘘) 后, 与 DOT1L-inhibitor 和二甲基亚砜控制和更新它每第二天 (比较 2.1.12)。如果需要, 通过标准免疫方法测试抑制剂处理的效率。对于芯片, 继续步骤3.3。 注意: 在盘子上留下 CGM (与 FBS) 将导致 CGNPs 分化为小脑颗粒神经元 (CGNs)。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 细胞固定和染色质剪切
注意: 如果单元格未被培养, 则执行步骤3.1 和 3.2, 如果它们正在进入步骤3.3。
<ol><li>通过离心收集细胞4分钟 1000 x g, 并添加1.3 毫升 PBS。将样品转移到1.5 毫升的管子。离心机为4分钟在 1000 x g 在4° c。用1.3 毫升 PBS 清洗样品两次。</li>
	<li>
关键步骤:添加350µL 1% 粉煤灰的样品和孵化5分钟, 在22° c。为了停止反应, 添加18.4 µL 甘氨酸和1毫升 PBS。离心样品5分钟在 1000 x g 在4° c。<ol>
<li>用冰冷 PBS 洗两次样品。用离心法收集固定细胞5分钟, 在 1000 x g 和4° c。从现在起把样品放在冰上。 注意: 固定时间必须精确5分钟才能获得最佳效果。不同的细胞数可能需要时间适应。</li>
	</ol>
</li>
	<li>
关键步骤:对培养的 CGNPs (或党), 添加多达1毫升1% 的粉煤灰直接到细胞培养板井。在22° c 的5分钟孵育后, 加入适量的甘氨酸和 PBS, 用刮板机收割细胞。<ol>
<li>将细胞转化为1.5 毫升的管子, 并将样品离心5分钟, 在 1000 x g 和4° c。用冰冷 PBS 洗两次样品。在4° c 的 5 minat 1000 x g 的离心收集固定细胞。从现在起把样品放在冰上。</li>
	</ol>
</li>
	<li>
关键步骤:加入700µL 的裂解缓冲液 (+ 蛋白酶抑制剂), 在4° c 下孵育样品15分钟。涡流样品每5分钟切变裂解细胞的染色质 3 x 10 min 在 sonicator (三十年代脉冲, 三十年代暂停) 在最大力量。<ol>
<li>确保每根管子的体积不超过350µL。每10分钟将样品涡旋, 用相衬显微镜检查残余核的裂解液。 注意: 获得最佳剪切效果 (与图 1B比较) 以便以后进行分析是很重要的。在使用其他方法来剪切染色质的情况下, 优化剪切到染色质碎片大小之间的 200-500 bp。</li>
	</ol>
</li>
	<li>颗粒细胞残余物为 10 min, 1.3万 x g, 4 ° c。使用上清 preclearing 步骤5.2。如有需要, 在-20 ° c 冻结样品以备日后使用。</li>
</ol>4. 珠子和 Preclearing 的制备
<ol><li>洗涤45µL 蛋白质磁珠/芯片和20µL 磁珠/样品与1毫升冰冷 PBS 含有0.02% 吐温三次使用磁性立场。然后, 加入1毫升的冰冷 PBS 的珠子。</li>
	<li>1毫升冰冷 PBS 和45µL 珠/芯片, 添加3µg 抗体/芯片 (H3K79me2 或兔 IgG)。在4° c 的转子上孵育样品以将抗体与念珠结合。根据抗体, 使用〜3µg 抗体/芯片。</li>
	<li>用1毫升冰冷 PBS (含0.02% 吐温) 清洗抗体结合珠三次。添加适当的珠体积 (开始体积的磁珠使用) 冰冷 PBS 的洗涤抗体耦合珠。</li>
	<li>添加600µL 稀释缓冲器和20µL 的洗涤磁珠的样品 preclearing (总体积1320µL) 和孵化2小时在4° c 的转子。使用磁性支架取出珠子。采取 5% (33 µL) 的裂解作为输入样本, 并冻结他们在-20 ° c。</li>
</ol>5. 染色质沉淀
<ol><li>将 precleared 提取物分成两个管子 (大约643.5 µL), 加入相同体积的稀释缓冲液和抗体结合珠 (45 µL/样品;H3K79me2 或兔 IgG)。将样品在4° c 的一夜之间孵化。</li>
	<li>用冰冷的芯片缓冲1、芯片缓冲器2和芯片缓冲器 3, 在一个旋转器的4° c 上分别洗涤10分钟的珠子。之后, 用 TE 缓冲器洗涤三次, 在4° c 的旋转器上5分钟。</li>
	<li>洗在室温下, 用洗脱缓冲液在1小时内以 1400 rpm 在振动筛上。</li>
	<li>添加10µg 核糖核酸 (1 毫克/毫升) 每片样品和5µg 到输入样品和孵化样品为30分钟在37° c (1400 rpm)。</li>
	<li>添加100µg 蛋白酶 K (20 µg/µL) 每片样品和50µg 每输入和孵化过夜在65° c 在 1400 rpm。</li>
</ol>6. 芯片样品的提纯
<ol><li>用 DNA 纯化试剂盒净化芯片和输入样品 (参见材料表)。当超过5µg 的 DNA 被期望时, 使用更多的纯化柱。洗样品与 2 x 15 µL 的 DNA 洗脱缓冲器提供的试剂盒。</li>
	<li>通过使用可视化的荧光和 fluorospectrometer (请参见材料表) 对样本进行量化 (使用1µL)。</li>
</ol>7. 通过 qPCR 分析芯片样品
<ol><li>对于 qPCR 分析的入门设计, 从集成基因组查看器 (IGV) 浏览器34中检索感兴趣的基因组序列。在一个基因的转录起始点周围设计底漆, 因为 H3K79me2 很可能位于那里。作为一个控制, 考虑非编码的基因组区域或区域大约 10 kb 上游的技术支助或 10 kb 下游的转录终端网站 (附加费)。<ol>
<li>使用 https://www.ncbi.nlm.nih.gov/tools/primer-blast/70 和 200 bp 的产品尺寸和60° c 的最佳温度预设生成底漆。为试验目的, 使用在表 1中列出的基因组区域Aft4、Ddit3、 Scd1、 Aft3、和Npm1的引物。</li>
	</ol>
</li>
	<li>测试新设计的底漆与以下 qPCR 程序, 主混合, 和退火温度梯度在58° c 和63° c 之间, 并且选择退火温度以最高的产品数量和质量。<ol>
<li>应用 DNA 凝胶电泳控制预期的产品尺寸。对于 qPCR 分析, 请使用实时 PCR 检测系统 (请参阅材料表)。</li>
		<li>准备主混合使用10µL dna 聚合酶主混合, 0.5 µL 底漆向前 (10 µM), 0.5 µL 底漆反向 (10 µM), 4 µL 核酸无水, 和5µL dna (1 ng/µL)。</li>
		<li>使用2步 qPCR 程序如下: 5 分钟在95° c, 50x (三十年代在95° c, 三十年代在58° c-63 ° c) 和变性梯度经常在4° c。</li>
	</ol>
</li>
	<li>创建一个稀释系列的基因组, 剪切, 纯化 DNA 样本 (2 ng/µL, 1 ng/µL, 0.5 ng/µL, 0.25 ng/µL, 和 0.125 ng/µL) 和使用5µL 的效率测试使用 qPCR。确定标准曲线的斜率, 并通过以下公式计算底漆的效率: 效率 (%) = (10^ (-1/倾斜)-1) * 100。<ol>
<li>使用底漆与效率在90% 和105% 之间在一个理想的事例, 或者至少以效率在85% 和115% 之间。</li>
	</ol>
</li>
	<li>为了分析芯片和输入样品, 应用 0.1-1 ng 芯片 dna 混合与分别向前和反向底漆, 核酸无水, 和 DNA 聚合酶主混合在20µL 的最终体积为每 qPCR 反应。</li>
	<li>确定芯片和输入样本的阈值周期 (ct), 并将样本 c 的t规范化为 ct输入的值以计算浓缩量 (% 输入), 并使用以下公式。使用从 IgG 控制中获得的 Ct值来确定背景级别。 % 输入 = 100-2^(范数. 输入−范数。芯片) 规范.输入 = Ct (输入) −日志2(稀释因子) 规范.芯片 = Ct (芯片) −日志2(稀释因子) 注意: 规范。= 规范化</li>
</ol>8. 通过测序分析芯片样品
<ol><li>将芯片样本 (输入和 immunoprecipitated DNA 样本) 转移到测序设施。</li>
	<li>要预先准备库, 请使用适当的库准备工具包 (请参阅材料表), 并将输入 dna 的 2 ng 和所有的 immunoprecipitated dna 转换为索引库, 用于下一代复合序列指定的平台 (请参见材料表)。</li>
	<li>
关键步骤:按照试剂盒的指示, 结扎测序适配器 (工作浓度为15µM), 以结束修复和 dA 尾 DNA 片段。对于测序适配器和 PCR 引物使用适当的寡 (参见材料表)。对于这一数量的输入 dna, 使用 6-9 PCR 周期来丰富适配器结扎的 dna 片段。 注: 通常情况下, 不需要对适配器的 DNA 进行大小选择。最好使用尽可能少的 pcr 周期, 因为许多 pcr 扩增周期导致 pcr 重复和高 GC 偏倚。</li>
	<li>对于一个最终的库检查, 采取0.5 µL 的总反应分析, 以可视化大小分布在一个适当的设备 (请参见材料表)。对于量化, 使用可视化染料结合 fluorospectrometer 或其他方法 (请参见材料表)。</li>
	<li>因为 H3K79me2 似乎是一个组蛋白修饰, 这是广泛分布在更大的染色体区域, 序列样品配对结束以读的长度 50 bp 并且以测序深度75宇达读。</li>
	<li>分析与 galaxy/单向弗赖堡服务器 (galaxy..。</li>
	<li>执行质量控制与获得的 ChIPseq 与 FastQC, 如果合适, 直接映射他们与 Bowtie2 版本 2.2.035有或不修剪。作为参考基因组组装使用 mm9 或最新版本的 mm10;作为参考注释 Ensembl FTP 版本79。</li>
	<li>可选: 在峰值呼叫前使用皮卡德 MarkDuplicates (http://broadinstitute.github.io/picard) 删除读取重复项。</li>
	<li>用 MACS2 版本 2.1.036调用峰值, 使用 H3K79me2 的 "宽" 选项。</li>
	<li>若要获取芯片和输入示例之间的日志2比率, 则两个示例的读取比率读取次数的日志2都使用 BamCoverage 和 BamCompare。</li>
	<li>对于所有其他深入的芯片-seq 特定分析, 应用 deepTools2 版本2.3.5 或 2.4.137,即生成覆盖跟踪文件 (仅用于芯片的 bamCoverage, bamCompare 用于芯片的比较), 估计芯片性能使用 plotFingerprint 和生成一般概述使用 computeMatrix, plotHeatmap。根据 H3K79me2 分布, 在 computeMatrix 过程中选择 k-均值聚类来聚类基因组区域。</li>
	<li>使用已发布的芯片 seq 数据作为 H3K79me2 的 E14.5 DT, 用于试验目的 (加入号码: SRP057733) NCBI。</li>
</ol>

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</ol>

作者宣布, 他们没有竞争的金融利益

有两种主要的方法来执行染色质沉淀, 以检测组蛋白的修饰, 转录因子的染色体的占有率, 蛋白的读者, 作家, 或橡皮擦。一种是用核酸消化的原生芯片法, 沉淀的原染色质, 另一种是采用粉煤灰固定的, 被剪切的染色质, 其中核和其他 dna 附着的蛋白质与 dna 共价键结合的方法。39. 本机芯片具有较高的抗体检测率, 应适用于组蛋白甲基化, 因为染色质和组蛋白 h3k4 在整个芯片中相对稳?...

大脑的感官、运动和认知功能非常复杂, 容易受到物理和环境变化的影响。大脑由后、中、前脑的三一般部分组成, 它们是紧密相连的。前脑内, 端可分为背部端 (DT) 和腹端 (VT)。小鼠的 DT 由六皮质层组成, 它们是在 E11.5 和 E18.5 之间形成的, 以 "内而外" 的方式1。VT 包括节主教在发育中, 后来形成基底节2,3。几种细胞类型可以归类在哺乳动物中枢神经系统, 如神经元, 星形胶质细胞, 或突4, 在颞空间的方式5发展。首先, 神经祖细胞 (npc) 产生不同类型的神经元, 神经元在 VT, 和投射神经元在 DT, 后来到神经胶质细胞 (例如, 星形细胞6)。在皮质发育过程中, 首先形成了含有卡哈尔 Retzius 细胞的最浅层 (I 层)。然后, 在 E12.5 和 E14.5 之间, npc 产生更深的神经元层 (VI, V), 而在14.5 和16.5 之间, 祖细胞引起上层 (IV-II) 神经元7,8。神经元的身份是由不同的形态诱导的颞空间转录程序和另外的后生程序2指定的。
小脑, 这是牵连在运动协调, 位于后脑和发展之间的 E10 和粗略 P20 在小鼠9。它包含小脑皮质和小脑核10。成人小脑皮质包括三层, 最外层的分子层, 浦肯野细胞层, 和包含颗粒状神经元的最里层的颗粒层10。小脑颗粒细胞是最小的神经元, 代表了脊椎动物大脑中大约80% 的神经元11。它们从位于外部生发区的前驱体发展而来, 并通过浦肯野细胞层迁移到它们的目标12。象在端, 小脑的发展由几个重要来说调控, 有具体时间和空间相关的作用和创始被定义的转录节目10。
皮质和小脑层的发育由特定来说的转录表达控制, 因此, 由 DNA 的染色质状态。在一个简化的观点, 染色质状态可以被划分成染色质作为转录活跃和异作为转录沈默区域。核作为染色质的基本单位, 包含了每个核心组蛋白 H2A、H2B、H3 和 H4 的两个拷贝, 周围有147碱基对 DNA13。组蛋白是高度后翻译修饰的甲基化, 乙酰化, 磷酸, 泛, sumo, ADP adp-, 脱, 和脯氨酸异构化反应14,15。组蛋白赖氨酸甲基化被认为是最稳定的蛋白修饰, 控制转录, 复制, 重组16, DNA 损伤的反应,17, 和基因印记18。Lysines 可以是单、di 或三甲基化的19 , 并且不仅出现在可访问的组蛋白尾部, 而且还显示在组组的球状域中20。具体的 h3k4 在 H3K4 和 H3K36 主要与染色质, 具体 h3k4 在 H3K9, H3K27, 或 H4K20 主要在异地区发现, 虽然所有残留物位于组蛋白尾部14, 19,21。H3K79 甲基化位于组蛋白球状域内, 并与转录活性有关, 但也与转录惰性基因的区域22有关。由于在酵母、小牛胸腺、鸡肉和人类23中观察到了这种改变, 因此在进化上是保守的。H3K79 单, di, 和 trimethylation (H3K79me1, me2, me3) 是由组蛋白甲基 DOT1L24,25和核集合域含蛋白质 2 (Nsd2)26。DOT1L 与增殖、DNA 修复和细胞 reprograming27有牵连。Dot1l在小鼠中的丢失导致在发育阶段 E10.528,29周围的产前死亡。在心脏发育和 myocardiocyte 分化期间, DOT1L 是基因表达调控的关键30。在中枢神经系统中, DOT1L 功能可能牵连神经管发育31, 它涉及在前脑发育32期间抑制Tbr1表达, 并可在 ER 应力调节中起作用响应基因33。H3K79me 的上下文相关激活或镇压行动, 特别是与在体内情况象中央神经系统的发展, 是到目前为止仅部份地被了解的32。由于 H3K79 甲基化位于组蛋白3的球状域中, 因此与灵活的组蛋白尾23的修饰相比, 阻的可访问性较差。为了了解 H3K79 甲基化的作用, 需要可靠和可重复的分析方法来确定其位置和基因组环境。在本方法中, 我们提出了不同的神经祖细胞的分离方法 (党用于小脑的皮质和 CGNPs), 有效的 DOT1L 抑制剂治疗, 以及在不同时间通过 qPCR 或测序来分析 H3K79 甲基化的芯片方法在皮质和小脑发育过程中的点。有关协议及其可能性的概述, 请参见图 1。

<table><tbody><tr><td>Anti-GAPDH</td><td>Abcam</td><td>ab8245</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:5000</td></tr><tr><td>Anti-H3</td><td>Abcam</td><td>ab1791</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:3000</td></tr><tr><td>Anti-H3K79me2</td><td>Diagenode</td><td>pAb-051-050</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; ChIP antibody</td></tr><tr><td>Anti-H3K79me2</td><td>Abcam</td><td>ab-051-050</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:1000</td></tr><tr><td>Anti-rabbit-IgG</td><td>Diagenode</td><td>C15410206</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; ChIP Ctrl antibody</td></tr><tr><td>Anti-Tubulin alpha</td><td>Abcam</td><td>ab108629</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:3000</td></tr><tr><td>Apo-Transferrin (1 mg/ml)</td><td>Sigma-Aldrich</td><td>T1147</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>B27 Supplement (50x)</td><td>Life Technologies</td><td>17504044</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Bioanalyzer</td><td>Agilent technologies</td><td>G2940CA</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For analysis of sheared chromatin</td></tr><tr><td>Bioruptor NextGen</td><td>Diagenode</td><td>B01020001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Ultrasonicator</td></tr><tr><td>Boric acid pH 8.4</td><td>Sigma Aldrich</td><td>B6768</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>CFX Connect RT PCR Detection System</td><td>Bio-Rad</td><td>1855201</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Detection system for qPCR</td></tr><tr><td>DMEM-F12</td><td>Life Technologies</td><td>11320-033</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGM</td></tr><tr><td>Dynabeads Protein A</td><td>Invitrogen</td><td>10001D</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Magnetic beads, for ChIP</td></tr><tr><td>EPZ-5676</td><td>Selleckchem</td><td>S7062</td><td>&lt;strong&gt;Category:&lt;/strong&gt; DOT1L inhibition&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For DOT1L inhibition in cell culture</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>SERVA</td><td>39760.01</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; EDTA</td></tr><tr><td>Fetal Bovine Serum 10% (v/v)</td><td>Gibco</td><td>10082147</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC isolation and CGM</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Glutathione (1.25 mg/ml)</td><td>Sigma-Aldrich</td><td>G4251</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3187</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For cell fixation</td></tr><tr><td>GoTaq mastermix</td><td>Promega</td><td>A6002</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; DNA polymerase master mix for qPCR</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025-100</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; HBSS</td></tr><tr><td>L-glutamine (200 mM)</td><td>Life Technologies</td><td>25030081</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>Lithium chloride</td><td>Sigma-Aldrich</td><td>L4408</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; LiCl</td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGM</td></tr><tr><td>NanoDrop 3300</td><td>Thermo Fisher</td><td>3300</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Fluorospectrometer for DNA quantification</td></tr><tr><td>NEB Next Ultra DNA Library Prep Kit for Illumina</td><td>NEB</td><td>E7645S</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Kit for Library preparation</td></tr><tr><td>NEBNext Multiplex Oligos for Illumina</td><td>NEB</td><td>E7335</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Oligos for Library preparation</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>NP-40 Alternative</td><td>Calbiochem</td><td>492016</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP buffer</td></tr><tr><td>Paraformaldehyde</td><td>Carl Roth</td><td>335</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PFA, for cell fixation</td></tr><tr><td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td><td>Life Technologies</td><td>15640055</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PSN, for CCM and CGM</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010023</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PBS, for CPC isolation</td></tr><tr><td>PicoGreen Kit</td><td>Thermo Fisher</td><td>P11496</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Visualizing dye for DNA quantification</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Poly-L-ornithine hydrobromide</td><td>Sigma Aldrich</td><td>P3655</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>Potassium chloride</td><td>Thermo Fisher</td><td>AM9640G</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; KCl, for CGM</td></tr><tr><td>Protease inhibitor</td><td>Roche</td><td>4693159001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>3115879001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Qiagen MinElute</td><td>Qiagen</td><td>28004</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Kit for DNA purification</td></tr><tr><td>RNAse</td><td>Sigma-Aldrich</td><td>R6513</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>SGC0946</td><td>Selleckchem</td><td>S7079</td><td>&lt;strong&gt;Category:&lt;/strong&gt; DOT1L inhibition&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For DOT1L inhibition in cell culture</td></tr><tr><td>Sodium bicarbonate</td><td>Carl Roth</td><td>8551.1</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; for Elution buffer</td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>9265</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; NaCl, for ChIP buffer</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>30970</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Sodium dodecylsulfate</td><td>Carl Roth</td><td>183</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; SDS, for ChIP</td></tr><tr><td>Sonic hedgehock (SHH)</td><td>Sigma-Aldrich</td><td>SRP6004</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Superoxide dismutase (1mg/ml)</td><td>Sigma-Aldrich</td><td>S7571</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Carl Roth</td><td>9090</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; TRIS, for ChIP buffer</td></tr><tr><td>Triton X-100</td><td>Carl Roth</td><td>X100</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP buffer</td></tr><tr><td>Trypsin-EDTA 0,05% (w/v)</td><td>Sigma Aldrich</td><td>59417C</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC isolation</td></tr><tr><td>Tween20</td><td>Carl Roth</td><td>28320</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For bead preparation</td></tr><tr><td>Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath</td><td></td><td></td><td></td></tr><tr><td>CCM: Cortical cell medium</td><td></td><td></td><td></td></tr><tr><td>CGM: CGNP cell culture medium</td><td></td><td></td><td></td></tr></tbody></table>

isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark

大脑发育是一个复杂的过程, 它是由来说的梯度和不同的转录程序控制在颞空间的方式。此外, 后生染色质修饰, 如组蛋白甲基化, 有一个重要的作用, 建立和维护特定的细胞命运在这个过程中。大多数组蛋白甲基化发生在灵活的组蛋白尾部, 这是组蛋白修饰剂、橡皮擦和组蛋白的阅读蛋白。相比之下, H3K79 甲基化位于组蛋白3的球状领域, 并与不同的发育功能有牵连。H3K79 甲基化是进化保守的, 可以从智人到酿酒酵母的种类繁多的物种中发现。改变发生在不同的细胞数量的生物体内, 包括神经祖。H3K79 甲基化在组蛋白3的球状域中的位置使得难以评估。在这里, 我们提出的方法分离和培养皮层祖细胞 (党) 从胚胎皮质脑组织 (E11.5-E14.5) 或小脑颗粒神经元祖 (CGNPs) 从产后组织 (P5-P7), 并有效 immunoprecipitateH3K79me2 定量 PCR (qPCR) 和全基因组测序。

神经祖分离、培养、H3K79me2 芯片和芯片分析方法的一般方案:图 1显示了一个流程图, 用于在胚胎发育期或产后阶段的小脑颗粒神经元祖细胞的不同时间点执行 H3K79me2 芯片。作为第一步, 大脑必须被孤立, 端 (在 E11.5 和 E14.5 之间) 或小脑 (P5-P7) 必须被检索。通过将端划分为 DT 和 VT, 可以分析不同区域。随后组织将匀浆和神经祖细胞分离。现?...

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Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

我们提出一种有效和可重复的方法, 分离和培养胚胎和产后脑组织中的神经祖细胞染色质沉淀 (芯片) 的组蛋白3赖氨酸 79 dimethylation (H3K79me2)-一个组蛋白标记位于组蛋白3的球状域。

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

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7.4K 次观看.  University of Freiburg. 我们提出一种有效和可重复的方法, 分离和培养胚胎和产后脑组织中的神经祖细胞染色质沉淀 (芯片) 的组蛋白3赖氨酸 79 dimethylation (H3K79me2)-一个组蛋白标记位于组蛋白3的球状域。

视频: Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

科研

发育生物学

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

生物工程

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

利用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 ...

癌症研究

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

本地化的蛋白质三维神经干细胞培养：一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

神经科学

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 Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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 ...

JoVE Encyclopedia of Experiments

Neuropathology

Tumors

Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

Source: Meyer, S. et al., <a href="https://app.jove.com/v/52831">Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

分离源自成人成纤维细胞的诱导神经祖细胞集落

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Putative Induced ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app.jove.com/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

分离和培养小鼠小脑颗粒神经元祖细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...