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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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>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:5000</td>
		</tr>
		<tr>
			<td>Anti-H3</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:3000</td>
		</tr>
		<tr>
			<td>Anti-H3K79me2</td>
			<td>Diagenode</td>
			<td>pAb-051-050</td>
			<td>Category: Antibody 
			Abbreviation/Comment: ChIP antibody</td>
		</tr>
		<tr>
			<td>Anti-H3K79me2</td>
			<td>Abcam</td>
			<td>ab-051-050</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:1000</td>
		</tr>
		<tr>
			<td>Anti-rabbit-IgG</td>
			<td>Diagenode</td>
			<td>C15410206</td>
			<td>Category: Antibody 
			Abbreviation/Comment: ChIP Ctrl antibody</td>
		</tr>
		<tr>
			<td>Anti-Tubulin alpha</td>
			<td>Abcam</td>
			<td>ab108629</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:3000</td>
		</tr>
		<tr>
			<td>Apo-Transferrin (1 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent technologies</td>
			<td>G2940CA</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For analysis of sheared chromatin</td>
		</tr>
		<tr>
			<td>Bioruptor NextGen</td>
			<td>Diagenode</td>
			<td>B01020001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Ultrasonicator</td>
		</tr>
		<tr>
			<td>Boric acid pH 8.4</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>CFX Connect RT PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855201</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Detection system for qPCR</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Invitrogen</td>
			<td>10001D</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Magnetic beads, for ChIP</td>
		</tr>
		<tr>
			<td>EPZ-5676</td>
			<td>Selleckchem</td>
			<td>S7062</td>
			<td>Category: DOT1L inhibition 
			Abbreviation/Comment: For DOT1L inhibition in cell culture</td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid</td>
			<td>SERVA</td>
			<td>39760.01</td>
			<td>Category: ChIP 
			Abbreviation/Comment: EDTA</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum 10% (v/v)</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation and CGM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Glutathione (1.25 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>G4251</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>3187</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For cell fixation</td>
		</tr>
		<tr>
			<td>GoTaq mastermix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: DNA polymerase master mix for qPCR</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14025-100</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: HBSS</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>L4408</td>
			<td>Category: ChIP 
			Abbreviation/Comment: LiCl</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>NanoDrop 3300</td>
			<td>Thermo Fisher</td>
			<td>3300</td>
			<td>Category: ChIP 
			Abbreviation/Comment: 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>Category: ChIP Analysis 
			Abbreviation/Comment: Kit for Library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos for Illumina</td>
			<td>NEB</td>
			<td>E7335</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Oligos for Library preparation</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>NP-40 Alternative</td>
			<td>Calbiochem</td>
			<td>492016</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP buffer</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>335</td>
			<td>Category: ChIP 
			Abbreviation/Comment: PFA, for cell fixation</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td>
			<td>Life Technologies</td>
			<td>15640055</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PSN, for CCM and CGM</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PBS, for CPC isolation</td>
		</tr>
		<tr>
			<td>PicoGreen Kit</td>
			<td>Thermo Fisher</td>
			<td>P11496</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Visualizing dye for DNA quantification</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Thermo Fisher</td>
			<td>AM9640G</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: KCl, for CGM</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>3115879001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Qiagen MinElute</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Kit for DNA purification</td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>SGC0946</td>
			<td>Selleckchem</td>
			<td>S7079</td>
			<td>Category: DOT1L inhibition 
			Abbreviation/Comment: For DOT1L inhibition in cell culture</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Carl Roth</td>
			<td>8551.1</td>
			<td>Category: ChIP 
			Abbreviation/Comment: for Elution buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265</td>
			<td>Category: ChIP 
			Abbreviation/Comment: NaCl, for ChIP buffer</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Sodium dodecylsulfate</td>
			<td>Carl Roth</td>
			<td>183</td>
			<td>Category: ChIP 
			Abbreviation/Comment: SDS, for ChIP</td>
		</tr>
		<tr>
			<td>Sonic hedgehock (SHH)</td>
			<td>Sigma-Aldrich</td>
			<td>SRP6004</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Superoxide dismutase (1mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>S7571</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Carl Roth</td>
			<td>9090</td>
			<td>Category: ChIP 
			Abbreviation/Comment: TRIS, for ChIP buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>X100</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0,05% (w/v)</td>
			<td>Sigma Aldrich</td>
			<td>59417C</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Carl Roth</td>
			<td>28320</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For bead preparation</td>
		</tr>
		<tr>
			<td colspan="4">Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath</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?...

Watch this Scientific Journal Video about Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark at JoVE.com

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

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

科研

神经科学

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

隔离和肉毒杆菌神经毒素定量从复杂的基质使用BoTest矩阵测定

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...