这项工作得到国家卫生研究院国立医学研究院的支持, 通过赠款 5P20GM103636-02 (包括流式细胞术核心支持) 和 1R01GM121703, 以及俄克拉荷马州成人干细胞中心颁发的奖项。研究。

所有动物都严格按照俄克拉荷马州医学研究基金会机构动物保育和使用委员会批准的协议处理。
1. 建立成年斑马鱼进行育种
<ol><li>使用一大群成年雄性和雌性斑马鱼的单一菌株进行育种。斑马鱼的基因组成有小的差异, 即使是单一的菌株, 使用一个大的队列, 以确保结果是代表的遗传变异的人口, 不限于一个小的子集的人口。</li>
	<li>在胚胎的前一天晚上将被收集, 安置许多繁殖的成人入饲养坦克, 使用大约相等的数量的男性和女性。根据实验, 使用下列育种策略之一。<ol>
<li>育种策略 1: 在单独的饲养池中放置一些雄性和雌性斑马鱼, 将雄性与雌性分隔开来。如果使用这种方法, 设置许多不同的繁殖池和池的胚胎, 以确保生物变异性是代表的人口。</li>
		<li>育种策略 2: 将许多雄性和雌性斑马鱼组合在一个大型繁殖池中。使用这个策略只要有足够多的胚胎, 它是合理的假设他们来自不同的创始人, 因此是人口的基因代表。</li>
	</ol>
</li>
</ol>2. 定时交配收集、分拣和住房斑马鱼胚胎进行实验
<ol><li>执行定时交配, 开始时, 光周期是在早上。丢弃任何一夜之间产生的胚胎。</li>
	<li>允许鱼在浅水中繁殖 (3-5 厘米), 一段时间为10分钟, 有一个假底供胚胎逃生。</li>
	<li>10分钟后, 通过过滤器倒入胚胎, 用洗涤瓶冲洗成10厘米厚的盘子。在同一时间点收集所有的胚胎, 标记他们与收集的时间, 并立即放置在一个孵化器在28.5 摄氏度。</li>
	<li>数以百计的胚胎可以期待从一个单一交配对一天。允许成年人在10分钟内繁殖, 直到获得的胚胎数量少于二十。</li>
	<li>约 1-1. 5 小时后收集, 排序胚胎, 以消除死和未受精的胚胎。对胚胎进行分类, 从孵化器中取出并在解剖显微镜下观察。<ol>
<li>计数的胚胎和地方密度为100胚胎每10厘米板。</li>
		<li>添加新鲜的 E3 培养基 (5 毫米氯化钠, 0.17 毫米氯化钾, 0.33 毫米 CaCl2, 0.33 毫米 MgSO4) 和返回胚胎到28.5 °c 孵化器。</li>
	</ol>
</li>
</ol>3. Dechorionate、deyolk 和修复斑马鱼胚胎
注: 本协议的这一部分是为 48 h 后受精 (hpf) 之前的胚胎设计的。在发育后期 (48 hpf 后), 没有必要去除胚胎的 chorions, 因为它们经常自然脱落。没有必要 deyolk 或删除 chorions 5 天后受精 (dpf) 的鱼。
<ol><li>当胚胎达到理想的发育年龄时, 根据 "斑马鱼的胚胎发育阶段"17, 在光学显微镜下验证适当的发育阶段。</li>
	<li>将多达1500级的胚胎转移到一个15毫升的锥形管上, 用 E3 洗几次。<ol>
<li>添加1毫升的 E3 和20µL 的链霉蛋白酶溶液 (30 毫克/毫升), 每100胚胎和轻轻的漩涡。可在单管中 dechorionated 1500 个胚胎, E3 15 毫升。</li>
		<li>放置管在其一侧, 并安排胚胎在一个均匀的层, 以确保最大的表面体积比。每2-3 分钟轻轻搅动管子, 直到所有 chorions 从胚胎中脱落。总 dechorionation 时间将因链霉蛋白酶活动而异。</li>
	</ol>
</li>
	<li>去除上清液, 用10毫升的 E3 轻轻冲洗胚胎3次。将胚胎放在冰上, 用于该议定书的其余部分。</li>
	<li>去除 E3 和洗涤胚胎用新鲜地被准备的冰冷 deyolking 缓冲 (0.5x 金兹堡鱼响铃的解答, 不用钙:55 毫米氯化钠, 1.8 毫米氯化钾, 1.25 毫米 NaHCO3)。<ol>
<li>添加1毫升的冰冷 deyolking 缓冲, 多达500个胚胎。</li>
		<li>使用 P1000, 轻轻地将胚胎移出5-10 次, 以扰乱蛋黄袋。</li>
		<li>转移1毫升到一个1.5 毫升离心管和离心机在4°c 和 500 x g 为5分钟。</li>
	</ol>
</li>
	<li>用1毫升的冰冷 1x PBS (磷酸盐缓冲盐水, pH 值 7.0) 除去上清和洗丸, 以消除响铃的解决方案。离心机在4°c 和 500 x g 为5分钟。</li>
	<li>小心去除1毫升 1x PBS 中的上清和并用重悬细胞。把管子放在冰上。</li>
	<li>将3毫升冰冷乙醇 (乙醇) 加入15毫升圆锥管。乙醇的涡流管轻轻地放在低的设置上。<ol>
<li>使用 P1000, 缓慢 (1 滴每秒) 滴斑马鱼胚胎细胞悬浮到乙醇, 而涡流轻轻。</li>
		<li>添加1毫升的胚胎细胞悬浮液, 用于最终固定溶液的75% 乙醇。</li>
	</ol>
</li>
	<li>在1毫升的细胞悬浮被添加后, 轻轻地旋流管混合和放置在-20 °c 至少1小时。建议将管放在他们的一侧, 以确保最大的表面体积比和减少的胚胎和细胞, 坚持在一起。储存胚胎细胞在-20 °c 2-3 周。</li>
</ol>4. DNA 的染色和对胚胎的分类
注: 本协议的这一部分是为 1 dpf 的胚胎设计的。
<ol><li>从-20 °c 和离心机中取出乙醇固定胚细胞, 4 °c 1500 x 克, 5 分钟。<ol>
<li>把管子放在冰上, 小心地除去上清。使用 P1000, 轻轻并用重悬细胞在1毫升新鲜制备的冷 pbs-BSA (1x pbs, 1% 牛血清白蛋白)。</li>
		<li>离心细胞在4°c 和 1500 x g 为5分钟和地方在冰。</li>
	</ol>
</li>
	<li>去除碘碘 (PI) 染色溶液中的上清和并用重悬细胞 (50 µg/毫升 PI, 100 µg/毫升 RNase A, PBS, pH 7.0), 每1000个胚胎使用200µL。<ol>
<li>允许细胞在 PI 溶液中孵化30分钟冰, 轻轻混合每5分钟。</li>
		<li>在冰上放置一个40µm 尼龙细胞过滤器在50毫升锥形管上。轻轻地吸管细胞混合和过滤通过网格。 注: 如果在该议定书3节中收集了来自同一 timepoint 的多个胚胎管, 则将这些胚胎结合在一起, 以便它们全部排列在一起。</li>
		<li>使用注射器柱塞内端的扁平, 轻轻地扰乱网格上剩余的细胞簇。</li>
		<li>通过过滤器冲洗细胞, 每1000胚胎500µL 冷 PBS。</li>
		<li>将40µm 网格放在15毫升的锥形管上, 然后将电池悬浮液从50毫升锥形管中再次过滤到15毫升管中。</li>
	</ol>
</li>
	<li>使用适当的细胞分选仪, 将激光激发到561纳米和发射检测到 610/20, 以检测碘碘化物。<ol>
<li>将15毫升的染色细胞涡流, 并在仪器中放置4摄氏度。</li>
		<li>以慢速运行细胞, 理想的1.0 毫升/分钟, 以获得一个干净的细胞周期剖面, 避免混合 G1 或 G2/M 细胞与 S 阶段人口。</li>
		<li>首先, 门为 FSC-一个 x SSC-a (向前散布区域由边散布区域) 去除残骸 (图 1A)。 注意: 从胚胎细胞可以不同的大小取决于阶段和细胞类型。中性密度 (ND) 滤波器可能需要在1.0 和1.5 之间切换, 具体取决于所呈现细胞的大小。大面积收集大部分细胞和清除碎片。</li>
		<li>第二, 通过 pi 区域 (pi a) 来去除碎片和单元格双峰 (图 1B) 的 ssc 区域 (ssc a) 的门。</li>
		<li>第三, 门为 ssc-A 由 ssc-W (宽度) 删除任何剩余的单元格双峰 (图 1C)。</li>
	</ol>
</li>
	<li>在 x 轴上的 y 轴和碘碘化区 (PI a) 上的直方图上显示门控种群。对于 24 hpf 胚胎, 这应该给出一个定型的细胞周期配置文件, 如图 1D所示。<ol>
<li>要对 S 相位填充进行排序, 请将 G1 峰值右侧的门设置为 G2 峰值的左侧, 包括 G1 和 G2 种群的最小数量。对于 24 hpf 胚胎, 这通常包括约10-15% 的封闭人口 (请参见图 1D)。</li>
		<li>对 G1 人口进行排序, 设置 G1 峰左侧的门, 而不是通过山顶的顶部。调整 G1 门的宽度尽可能窄 (请参见图 1D)。</li>
		<li>后门的 G1 和 S 阶段的人口, 以确保正确的初始浇口 (图 1E)。</li>
	</ol>
</li>
	<li>将 G1 和 S 相种群分为15毫升圆锥管和3毫升的 PBS-BSA。目标应该是在每分数 (G1 和 S 阶段) 之间获得50万到100万个或更多的排序单元。</li>
	<li>分拣完成后, 离心管在 1500 x g 和4°c 为10分钟。 注: 小细胞颗粒将很难看到, 它是重要的, 以避免扰乱它, 所以最好使用一个摆动斗转子在固定角度的转子。</li>
	<li>小心地清除上清和并用重悬丸与1毫升 1x PBS。将细胞转移到1.5 毫升离心管和离心机在 6000 x g 和4°c 5 分钟。</li>
	<li>小心地将所有液体从管子中取出, 冷冻干颗粒在-20 摄氏度。将颗粒贮存在-20 摄氏度, 2-3 周。</li>
</ol>5. dna 分离、RNase 治疗和 dna 纯化
<ol><li>从-20 摄氏度移除细胞颗粒并放置在冰上。<ol>
<li>添加400µL 的 SDS 缓冲 (50 毫米三盐酸 (pH 8.0), 10 毫米 EDTA, 1 米氯化钠, 0.5% SDS) 的颗粒和并用重悬。</li>
		<li>添加蛋白酶 K 到最后浓度的0.2 毫克/毫升和混合涡流简要。</li>
		<li>孵育样品在56°c 为2小时, 漩涡每15分钟。</li>
	</ol>
</li>
	<li>2小时后, 通过添加400µL 苯酚-氯仿 (Phenol:Chloroform:Isoamyl 醇 25:24:1, 饱和10毫米三, pH 8.0, 1mM EDTA), 进行苯酚-氯仿萃取。<ol>
<li>漩涡为二十年代和离心机样品在 1.6万 x g 为5分钟分离阶段。</li>
		<li>小心移除上水相 (400 µL) 和转移到1.5 毫升管。</li>
	</ol>
</li>
	<li>通过添加40µL 醋酸钠 (pH 5.2)、2µL 糖原和1毫升乙醇来进行乙醇沉淀。<ol>
<li>漩涡彻底混合, 并在-20 °c 过夜, 以沉淀 DNA。样品也可以沉淀在-80 摄氏度或干冰上1小时。</li>
		<li>离心机样品在4°c 和 1.6万 x g 为30分钟为颗粒脱氧核糖核酸。</li>
		<li>用1毫升70% 乙醇, 丢弃上清和洗涤颗粒。离心机在4°c 和 1.6万 x g 为5分钟。</li>
		<li>在常温下丢弃上清液和风干颗粒2-3 分钟。</li>
	</ol>
</li>
	<li>在96µL 蒸压水中溶解颗粒, 加入4µL RNase A (2 毫克/毫升)。<ol>
<li>混合涡流和孵化在37°c 1 小时。</li>
	</ol>
</li>
	<li>在 5.2.1-5.2.2 步骤中加入100µL 苯酚-氯仿和以下过程, 进行苯酚-氯仿萃取。</li>
	<li>通过添加10µL 醋酸钠、1µL 糖原和250µL 乙醇, 并按照步骤 5.3.1 5.3.5 的过程执行乙醇沉淀。</li>
	<li>并用重悬颗粒在60µL 的 TE 缓冲器 (10 毫米三盐酸 (pH 8.0), 10 毫米 EDTA (pH8.0)) 和确定 DNA 浓度的定量荧光方法。储存 DNA 在-20 摄氏度。 注意: 使用基于荧光的 dna 结合染料, 或其他比基于紫外线光谱仪的方法更可靠的方式来量化 dna 是很重要的。</li>
</ol>6. DNA 文库的制备和下一代测序
注: 每种测序运行 (即, 突变体, 转基因, 细胞系等) 都需要每个生物源的 G1 拷贝编号参考样本。将相同的生物源中的所有 S 相样本与相同的测序运行到相同的 G1 参考。至少运行两个样本的生物复制, 以确保样本之间的一致性。
<ol><li>稀释1µg 的 DNA 到最后的体积50µL 和转移到专门管超声波。</li>
	<li>根据制造商的规格和协议, 在聚焦的 ultrasonicator 中, 250-300 bp 靶的剪切基因组 DNA。</li>
	<li>根据制造商的规格和协议, 使用自动电泳机验证剪切基因组 DNA 的质量和大小。确保被剪切的基因组 DNA 有一个大的峰值为 250-300 bp。</li>
	<li>根据制造商的协议, 在 Illumina 平台上, 使用 DNA 库准备套件和多重寡核苷酸来准备排序库。</li>
	<li>根据制造商的协议, 使用自动电泳机验证图书馆准备的质量。确保有一个大的峰值 400-450 bp, 并有最低峰值的底漆脂肪酸 (80-85 bp) 和适配器脂肪酸 (120 bp)。</li>
	<li>根据制造商的协议, 量化库使用 dna 库量化试剂盒进行下一代 dna 测序。</li>
	<li>将15µL 的库稀释为 5 nM 的浓度, 并根据需要合并复用库, 以实现每个示例所需的唯一映射读取计数。 注意: 每个示例的唯一映射读取数将决定该分辨率。使用500万的映射读取来评估斑马鱼基因组的复制时间。建议以10-20 元的读数开始。</li>
	<li>根据制造商的指示, 在下一代测序平台上执行配对端 100 bp 全基因组 DNA 测序。</li>
</ol>7. 测序数据分析
注: 本节中的说明旨在作为分析的指南。使用其他方法来对齐、筛选、处理等进行排序。议定书的这一部分将处理这项工作中的首选分析方法。如果使用其他方法, 请调整参数和函数以适应这些目的。下面的命令是在 Ubutnu 或 Mac 终端中输入的, 安装了相应的软件包。
<ol><li>从 UCSC 基因组浏览器中获取最新版本的斑马鱼基因组 (danRer10, 从该协议编写时), 使用以下命令: wget ftp://hgdownload.cse.ucsc.edu/goldenPath/danRer10/bigZips/danRer10.fa.gz-O danRer10.fa.gz<ol>
<li>使用以下命令解压缩 fa 文件: gunzip danRer10.fa.gz</li>
		<li>使用以下命令将序列数据与基因组 BWA: bwa 8 danRer10 read_1 fastq read_2 fastq > 产量. 萨姆</li>
	</ol>
</li>
	<li>使用带有以下命令的 samtools 将. sam 文件转换为. bam 文件: samtools 视图-bS 输入. sam > 输出. bam<ol>
<li>使用 samtools 与以下命令排序对齐的排序文件: samtools 排序输出. bam > output_sorted. bam</li>
		<li>使用 samtools 与以下命令一起索引. bam 文件: samtools 索引输出. bam</li>
	</ol>
</li>
	<li>使用以下命令标记 PCR 重复项: Xmx2g 罐 MarkDuplicates \ 输入 = 输入. bam 输出 = 输出. bam REMOVE_DUPLICATES = false METRICS_FILE = output_metrics .txt</li>
	<li>使用以下命令为每个染色体生成文本 (.txt) 读取文件: 为 i 在 {1.25};做铬 = "人权中心" $i;回声 $CHROM;samtools 视图输入. bam $CHROM |剪切 f 24、57、89 > readsChr $ {i} .txt;做 注意: 结果 .txt 文件中的列分别为标志、位置、mapQ、对中、对位置和片段大小。</li>
	<li>使用 textscan 函数将 .txt 文件读入 Matlab (或任何其他类似的软件)。<ol>
<li>通过设置 mapQ 阈值 30, 筛选出低映射质量读数。</li>
		<li>移除带有标志的读取, 而不是主对齐、PCR 重复或对映射到不同的染色体。</li>
		<li>过滤掉任何读与他们的对超出距离门限 2000年 bp。</li>
		<li>评估结果读取的统计信息, 以确定读取统计信息、覆盖率、插入大小和质量 (图 2) 的数量。</li>
	</ol>
</li>
	<li>通过计算每条染色体长度的 100 bp 间隔中的读数数, 确定每个样本的100个 bp 固定窗口中的读覆盖率。<ol>
<li>计算所有窗口的中间读计数。</li>
		<li>通过从中值中删除大于5标准偏差的窗口, 筛选出高覆盖率区域。</li>
	</ol>
</li>
	<li>通过在每个染色体上查找片段, 并使用滑动窗口200读取, 定义 G1 参考样本中分析的区域。<ol>
<li>通过计算为伴随的 G1 引用定义的200读取计数窗口中的 s 相位读取数, 确定每个 s 阶段示例中的读深度。</li>
	</ol>
</li>
	<li>使用软件中的 csaps 函数平滑原始数据, 其中的插值因子 (p) 为 10-16。</li>
	<li>将平滑数据正常化为 z 分数, 其平均值为 0, 标准偏差为1。</li>
</ol>

<ol>
	<li>Rhind, N., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing.">DNA replication timing.</a> Cold Spring Harb Perspect Biol. 5 (8), a010132 (2013).</li><li>Pope, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topologically+associating+domains+are+stable+units+of+replication-timing+regulation.">Topologically associating domains are stable units of replication-timing regulation.</a> Nature. 515 (7527), 402-405 (2014).</li><li>Rivera-Mulia, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+replication+timing+and+gene+expression+during+lineage+specification+of+human+pluripotent+stem+cells.">Dynamic changes in replication timing and gene expression during lineage specification of human pluripotent stem cells.</a> Genome Res. 25 (8), 1091-1103 (2015).</li><li>Hiratani, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+reorganization+of+replication+domains+during+embryonic+stem+cell+differentiation.">Global reorganization of replication domains during embryonic stem cell differentiation.</a> PLoS Biol. 6 (10), e245 (2008).</li><li>Hiratani, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+dynamics+of+replication+timing+revealed+by+in+vitro+models+of+mouse+embryogenesis.">Genome-wide dynamics of replication timing revealed by in vitro models of mouse embryogenesis.</a> Genome Res. 20 (2), 155-169 (2010).</li><li>Koren, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+relationship+of+DNA+replication+timing+to+different+forms+of+human+mutation+and+variation.">Differential relationship of DNA replication timing to different forms of human mutation and variation.</a> Am J Hum Genet. 91 (6), 1033-1040 (2012).</li><li>Ryba, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+developmental+control+of+replication-timing+domains+in+pediatric+acute+lymphoblastic+leukemia.">Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia.</a> Genome Res. 22 (10), 1833-1844 (2012).</li><li>Sima, J., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+correlations:+replication+timing+and+mutational+landscapes+during+cancer+and+genome+evolution.">Complex correlations: replication timing and mutational landscapes during cancer and genome evolution.</a> Curr Opin Genet Dev. 25, 93-100 (2014).</li><li>Veldman, M. B., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+developmental+model+organism+for+pediatric+research.">Zebrafish as a developmental model organism for pediatric research.</a> Pediatr Res. 64 (5), 470-476 (2008).</li><li>Link, B. A., Megason, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+Model+for+Development.">Zebrafish as a Model for Development.</a> Sourcebook of Models for Biomedical Research. , 103-112 (2008).</li><li>Siefert, J. C., Clowdus, E. A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+in+the+early+embryonic+development+of+aquatic+animal+species.">Cell cycle control in the early embryonic development of aquatic animal species.</a> Comp Biochem Physiol C Toxicol Pharmacol. 178, 8-15 (2015).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicol Sci. 86 (1), 6-19 (2005).</li><li>Dooley, K., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+human+disease.">Zebrafish: a model system for the study of human disease.</a> Curr Opin Genet Dev. 10 (3), 252-256 (2000).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> J Clin Invest. 122 (7), 2337-2343 (2012).</li><li>Siefert, J. C., Georgescu, C., Wren, J. D., Koren, A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+timing+during+development+anticipates+transcriptional+programs+and+parallels+enhancer+activation.">DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation.</a> Genome Res. 27 (8), 1406-1416 (2017).</li><li>Koren, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+human+DNA+replication+timing.">Genetic variation in human DNA replication timing.</a> Cell. 159 (5), 1015-1026 (2014).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li></ol>

作者没有什么可透露的。

斑马鱼提供了一个新的和独特的体内模型系统来研究 DNA 复制的时间。在这个实验性协议中, 当定时交配执行时, 可以在一天内收集数以千计的胚胎进行实验。这些胚胎通过精确定时和明显特征的发育阶段同步发展。斑马鱼可以很容易和准确地通过形态学使用显微镜, 因为斑马鱼胚胎外部发展, 并在光学上清晰。本议定书详细说明了斑马鱼胚胎在整个脊椎动物发育过程中的复制时机研究。
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为了成功地分裂细胞, 他们必须首先准确和忠实地复制他们的整个基因组。基因组复制以可重现的模式出现, 称为 DNA 复制定时程序1。DNA 复制定时与染色质组织、表观遗传学标记和基因表达式2,3相关。复制定时的变化发生在整个开发过程中, 与转录程序和对染色质标记和组织4、5的更改有显著关系。此外, 复制定时与突变频率相关, 并且在不同类型的癌症中观察到时间的变化6,7,8。尽管有这些观察, 复制时机建立和管制的机制和决定因素仍然很不清楚, 它在发展和疾病中所起的作用是未定的。此外, 直到最近, 整个脊椎动物发展过程中的全基因组复制定时变化才在细胞培养模型中进行了研究。
斑马鱼,斑马斑马, 非常适合在开发过程中研究复制定时在体内, 因为单个交配对可以产生数以百计的胚胎, 与哺乳动物开发的许多相似之处迅速发展9, 10。此外, 在斑马鱼的发展, 细胞周期, 染色质组织和转录程序的变化, 共享与 DNA 复制定时11。斑马鱼也是一个优秀的遗传模型, 因为它们特别容易被转基因、突变和目标突变所操纵, 而基因筛发现了脊椎动物发育所需的许多基因12。因此, 斑马鱼可以用来识别复制定时建立和维护的基因, 并观察解除管制复制时间对脊椎动物发育的影响。转基因线也可以用来评估复制时间从个别细胞类型分离在不同的发展 timepoints 或疾病的情况。重要的是, 有各种各样的人类疾病的斑马鱼模型, 可以用来调查复制定时在疾病形成和进展的作用9,13,14。
最近, 第一个复制定时配置文件是由斑马鱼生成的, 将其建立为研究复制定时在体内15中的模型系统。为了实现这一目标, 在发育的多个阶段和与成年斑马鱼分离的细胞类型中, 收集了斑马鱼胚胎的细胞。然后根据 DNA 含量对细胞进行分类, 以分离 G1 和 S 相种群。使用 G1 示例作为副本编号控件, 确定了 S 相位填充的复制数变化, 并用于推断相对复制计时16。然后可以直接比较不同的开发样本和单元格类型之间的复制时间变化, 这用于确定在脊椎动物开发过程中体内发生的复制定时的变化。这种方法比其他基因组方法具有多种优势, 主要是不需要用胸苷类似物或 DNA 的免疫沉淀标记 4, 6.
这里详细介绍了在斑马鱼的高分辨率下对全基因组 DNA 复制时间进行描述的协议。这些协议用于确定斑马鱼基因组和表观遗传特征之间的关系, 以及在整个开发过程中发生的这些关系的变化。这些协议也很容易适应研究斑马鱼突变株和疾病模型中复制时间的变化。此外, 这些方法还提供了一个基础, 可以扩展到研究特定细胞类型的复制时间, 首先从斑马鱼的个别细胞类型排序。斑马鱼可以作为一个优秀的体内模型系统来研究复制时间, 并最终揭示这种重要的表观遗传特征的生物学功能。

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>EMD Millipore</td>
			<td>MMX00701</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>10165921001</td>
			<td>protease solution</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>KOPTEC</td>
			<td>V1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9647-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide (PI)</td>
			<td>Invitrogen</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9844</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Santa Cruz</td>
			<td>sc-24950</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform</td>
			<td>Sigma</td>
			<td>P3803-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>J.T.Baker</td>
			<td>3470</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Ambion</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Quanit-iT</td>
			<td>Invitrogen</td>
			<td>Q33130</td>
			<td>Reagents for fluorescence-based DNA quantification</td>
		</tr>
		<tr>
			<td>Covaris AFA microTUBE</td>
			<td>Covaris</td>
			<td>520045</td>
			<td>specialized tube for sonication</td>
		</tr>
		<tr>
			<td>Covaris E220 Sonicator</td>
			<td>Covaris</td>
			<td>E220</td>
			<td>focused ultrasonicator</td>
		</tr>
		<tr>
			<td>Agilent 4200 Tapestation</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td>automated electrophoresis machine</td>
		</tr>
		<tr>
			<td>D1000 ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td>Reagents for automated electrophoresis machine</td>
		</tr>
		<tr>
			<td>NEBNext Ultra DNA Library Prep Kit for Illumina</td>
			<td>NEB</td>
			<td>Cat#E7370L</td>
			<td>DNA library preparation kit</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos Kit for Illumina (Index Primers Set 1)</td>
			<td>NEB</td>
			<td>Cat#E7335S</td>
			<td>multiplex oligos for DNA library preparation kit</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos Kit for Illumina (Index Primers Set 2)</td>
			<td>NEB</td>
			<td>Cat#E7500S</td>
			<td>additional multiplex oligos for DNA library preparation kit</td>
		</tr>
		<tr>
			<td>NEBNext Library Quant Kit for Illumina</td>
			<td>NEB</td>
			<td>E7630L</td>
			<td>quantification kit for library preparation</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>magnetic beads</td>
		</tr>
		<tr>
			<td>Illumina HiSeq 2500</td>
			<td>Illumina</td>
			<td>SY&#8211;401&#8211;2501</td>
			<td>next generation DNA sequencing platform</td>
		</tr>
		<tr>
			<td>40 &#181;m Falcon Nylon Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Disposable Petri Dish 100 x 25 mm</td>
			<td>VWR</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 mL Syringe for Nichiryo Model 8100</td>
			<td>VWR</td>
			<td>89078-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click Tubes, 1.7 mL, Natural Color</td>
			<td>Denville Scientific</td>
			<td>C2170 (1001002)</td>
			<td>Dnase/Rnase free</td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Bottles</td>
			<td>VWR</td>
			<td>16650-022</td>
			<td>Low-Density Polyethylene, Wide Mouth</td>
		</tr>
		<tr>
			<td>Strainer</td>
			<td>VWR</td>
			<td>470092-440</td>
			<td>6.9 cm, fine mesh</td>
		</tr>
		<tr>
			<td>Corssing tank</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td>individual breeding tank</td>
		</tr>
		<tr>
			<td>iSpawn</td>
			<td>Techniplast</td>
			<td>N/A</td>
			<td>large breeding tank</td>
		</tr>
		<tr>
			<td>FACSAria II</td>
			<td>BD biosciences</td>
			<td>N/A</td>
			<td>cell sorting machine</td>
		</tr>
		<tr>
			<td>Wild M5a steromicroscope</td>
			<td>Wild Heerbrugg</td>
			<td>N/A</td>
			<td>dissecting microscope</td>
		</tr>
		<tr>
			<td>Qubit 3 Fluorometer</td>
			<td>Thermo Scientific</td>
			<td>Q33216</td>
			<td>quantitative fluorescence-based method for determining DNA concentration</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>version 2017a</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab Statistics Toolbox</td>
			<td>Mathworks</td>
			<td>version 11.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab Curve Fitting Toolbox</td>
			<td>Mathworks</td>
			<td>version 3.5.5</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling dna replication timing using zebrafish as an in vivo model system

dna 复制定时是一种重要的细胞特征, 与染色质结构、转录和 DNA 突变率有显著的关系。复制时间的变化发生在开发和癌症的过程中, 但是复制定时在发展和疾病中的作用是未知的。斑马鱼最近建立了作为一个体内模型系统来研究复制时间。这里详细介绍了使用斑马鱼确定 DNA 复制定时的协议。在对胚胎和斑马鱼的细胞进行分类后, 通过对下一代测序数据的分析, 可以通过确定 dna 拷贝数的变化来构建高分辨率的全基因组 dna 复制定时模式。斑马鱼模型系统允许评估在整个开发过程中发生在体内的复制定时变化, 也可以用来评估单个细胞类型、疾病模型或变异线的变化。这些方法将有助于研究在发展过程中复制定时的建立和维护的机制和决定因素, 复制时间在突变和肿瘤发生中的作用, 以及扰动的影响。发展和疾病的复制时间。

使用已发布的复制定时数据, 提供了具有代表性的复制定时配置文件和质量控制措施15。最初的处理步骤包括将测序数据与基因组进行校准, 计算读出长度和基因组覆盖率统计, 过滤低质量、配对和 PCR 重复读数。在图 2中显示了典型的斑马鱼排序示例的读取统计信息。筛选后, 读取计数在可变大小的窗口中确定, 数据被平滑和规范化。...

Watch this Scientific Journal Video about Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System at JoVE.com

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

斑马鱼最近被用作一个体内模型系统, 用于研究开发过程中 DNA 复制的时机。这里详细介绍了使用斑马鱼胚胎来描述复制时间的协议。该协议可以很容易地适应于研究突变体、个体细胞类型、疾病模型和其他物种的复制时间。

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

7.8K 次观看.  Oklahoma Medical Research Foundation. 斑马鱼最近被用作一个体内模型系统, 用于研究开发过程中 DNA 复制的时机。这里详细介绍了使用斑马鱼胚胎来描述复制时间的协议。该协议可以很容易地适应于研究突变体、个体细胞类型、疾病模型和其他物种的复制时间。

视频: Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...