这项工作是支持的，部分由北卡罗莱纳大学夏洛特，美联银行基金优秀教师，并授予NIGMS（R15GM101571）提供资金。

1。 LSE准备<ol><li>集蛋，雌蛙（ 非洲爪蟾 ）注射两次。第一次注射（启动）为100 U PMSG（孕马血清促性腺激素）每青蛙。青蛙必须催芽前至少两天诱导产卵和引物的青蛙可用于长达两个星期。黄金青蛙，用3毫升注射器和27 G针在背淋巴囊皮下注射PMSG。 </li><li>诱导产卵，注入500 U HCG（人绒毛膜促性腺激素）每底漆青蛙在使用27 G针背淋巴囊皮下。孵育注入青蛙在不同的桶含2升1个马克的改性林格氏液（MMR）。允许青蛙产卵14-20小时之前收集。 </li><li>从桶中取出青蛙，倒出MMR解决方案，直到剩下大约100毫升。鸡蛋从水桶，将鸡蛋，250毫升的烧杯中。 </li><li>通过添加100毫升的2％半胱氨酸（调整dejelly蛋pH值至7.8，用10M KOH）。轻轻旋转一个倒置的玻璃巴斯德移液管（直径0.7厘米），大约每30秒的鸡蛋。弃去，更换新鲜半胱氨酸两次孵化过程中。 dejellying过程是在约5-15分钟完成鸡蛋时，在烧杯的底部形成了一个更简明的层。 </li><li>丢弃的半胱氨酸溶液和，洗鸡蛋3倍与0.25倍MMR。涡流的鸡蛋放在一个玻璃吸管的解决方案。 "坏"蛋是由简单的目视检查，并在外观上的"浮肿"白。 "坏"的鸡蛋会堆积在烧杯中后，旋转的中心。删除"坏"蛋的巴斯德吸管。 </li><li>洗蛋与蛋裂解缓冲液（ELB）的三倍。删除任何额外的"坏"蛋的巴斯德吸管。倒入鸡蛋液成14毫升的Falcon管。 </li><li>旋转Falcon管中，为55秒，在188×g离心（1100转）使用CL2 IEC临床台式离心机转子的摆动压缩鸡蛋。删除多余的缓冲区中蛋层的顶部。加入0.5μL抑肽酶/亮肽素股票和0.5微升的细胞松弛素B股每毫升夯实鸡蛋。 </li><li>离心机的鸡蛋在16500 XG（10,000 RPM）为15分钟，在4°C，使用在Sorvall RC6加超高速离心机HB6摆动转子。离心分离后，鸡蛋是在管内分离成三层:脂质，提取物，和蛋黄/颜料从顶部到底部，分别。穿刺的Falcon管中的中间提取物层具有21 G针的下部侧。小心地取出针的穿刺针可能会阻碍塑料。插入一个新的21 G针1毫升注射器穿刺部位收集提取物。慢慢地提取到注射器吸出，以避免气泡和污染的脂肪和蛋黄/颜料层。 </li><li>将提取到的冷冻1.5ml离心管。每毫升卵提取物，添加以下解决方案中的化学股票dicated终浓度（在括号中所示）:（1）10微升的放线菌酮（100微克/毫升），（2）1微升抑肽酶/亮抑酶肽（10μg/ ml的每个），（3）1微升的细胞松弛素B（5微克/毫升），（4）1微升二硫苏糖醇（1mM）的，以及（5）0.33微升诺考达唑（3微克/毫升）。反转的鸡蛋轻轻地提取至少10倍。这是的LSE，必须作出新鲜和4小时内使用。 4小时后或冻融破坏的质量LSE。 </li></ol> 2。精子染色质DNA损伤的方法治疗<ol><li>准备正常精子染色质，根据先前描述的方法13。 </li><li>准备MMS处理过的精子染色质<ol><li>正常的精子染色质重悬在0.5毫升缓冲十</li><li>加入500μL的悬浮染色质终浓度为100毫米〜5.5μl的MMS（9.1米股票）。 </li><li>与旋转的30分钟，在微量离心管中，在室温下孵育精子染色质。 </li><li>旋转米在686的XG（2,100转每分钟）10分钟，在室温下使用的IEC CL2临床离心机摆动转子和管接头的icrocentrifuge管。 </li><li>弃上清，将沉淀重悬于0.5毫升缓冲X加BSA（3％），DTT（0.1毫米）和抑肽酶/亮肽素（10微克/毫升）。 </li><li>重复步骤（4）和（5）两次洗精子染色质。 </li><li>确定用血球计数仪，精子染色质的浓度，并稀释至100,000精子个/μl。将5微升等份，在-80℃冰箱为进一步利用。 </li></ol></li><li>准备UV处理过的精子染色质<ol><li>加入所需量（ 例如，10微升）正常精子染色质石蜡膜一块的表面上。 </li><li>将封口膜UV交联成。设置所需的能量参数，根据你的实验，并开始损害精子染色质通过UV光。例如，它需要约21秒内达到1000焦耳/米2。 </li><li> UV辐照后应该被添加到卵提取物立即diation，UV处理的精子染色质。 </li></ol></li></ol> 3。制备的DNA损伤模仿结构（AT70） <ol><li>溶解两个合成的寡核苷酸的聚（dA的）70（指定为A70）和聚-（dT）的70（指定为T70），在水中的浓度为2微克/微升，分别。 </li><li>加入100μl的A70和T70的100微升至1.5 ml离心管中。煮沸混合的合成寡核苷酸的5分钟，在95℃在一个heatblock溶液。 </li><li>以干浴块（AT混合管），并让它冷却至室温实验室的长椅上。温度调整大约需要45-60分钟。 </li><li>将冷却的混合物是AT70与最终浓度为2微克/微升。存储10微升的AT70等分在-20°C冷冻室为进一步利用。 </li></ol> 4。在LSE损坏的精子染色质的DNA损伤引发的DNA损伤检验点模仿结构<ol><li>在LSE受损的精子染色质诱导DNA损伤检验点<ol><li>加入50微升至1.5 mL离心管中，LSE和1μl的能量合剂，受损的精子染色质（终浓度〜4,000精子/μl反应体系）和2μl补充。 </li><li>反应管中，在室温下孵育90分钟，轻弹管每10分钟。 </li><li>分送到显微镜载玻片辅以核染料溶液中30分钟温育后用1微升1微升的反应混合物。在反应混合物放置一个盖玻片和晶核形成通过荧光显微镜检查。通常情况下，核圆形，形成孵化30分钟后，启动DNA复制。 </li><li>入90μl的样品缓冲液中，以10微升的反应混合物。样品通过使用抗Chk1的P-S344中或反Chk1的抗体的免疫印迹分析。 </li></ol></li><li>在LSE与诱导DNA损伤检验点AT70 <ol><li>加入50微升至1.5 mL离心管中补充能量合剂和1微升1.6μl的变构股票LSE。 </li><li>将1.25微升预先制作AT70（2微克/微升）或水（作为阴性对照）添加到每个反应。反应在室温下孵育90分钟。滑动的反应管，每隔10分钟。 </li><li>入90μl的样品缓冲液中的反应混合物中加入10μl。通过免疫印迹使用抗Chk1的P-S344中或反Chk1的抗体的样品进行检查。 </li></ol></li></ol> 5。代表性的成果损坏的精子染色质或DNA损伤模仿结构可以触发ATR/Chk1-mediated的DNA损伤检查点，在非洲爪蟾卵提取物系统。 图2A表明，彩信诱导Chk1磷酸化在Ser344（Chk1的P-S344），这是一个指标ATR激酶的活化。 图2B示出，AT70，作为DNA损伤模仿STRU王国芳，也触发Chk1磷酸化。在这两个例子中加载控件作为总Chk1的样品。 <img alt="图1" src="/files/ftp_upload/4449/4449fig1.jpg" /> 图1。阿图的DNA损伤检查点的信号。 <img alt="图2" src="/files/ftp_upload/4449/4449fig2.jpg" /> 图2。 Chk1磷酸化引起的MMS或AT70在非洲爪蟾卵提取物治疗。（A）彩信（MMS）受损的精子染色质或正常的精子染色质（CON）卵提取物孵育90分钟。 Chk1的磷酸化Ser344（Chk1的P-S344），总Chk1在卵提取物，通过免疫检查。 （B）AT70或水（CON），加入鸡蛋提取物，分别。样品也进行了分析，通过免疫印迹法（A）中。

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没有利益冲突的声明。

在研究非洲爪蟾卵提取物的DNA损伤检验点有以下几个优点。卵提取物的使用提供了一个大的数量的无细胞提取物在相间的细胞周期同步。卵提取物，可以容易且廉价地作出。这是比较容易损坏的DNA或染色质，并揭示后从卵提取物immunodepleting目标蛋白在DNA损伤检验点缺陷。随后，一个潜在的功能缺损可以"救出"addback的野生型或突变型重组蛋白质。因此， 非洲爪蟾卵提取物是一种功能强...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>反Chk1的P-S344抗体</td><td>细胞信号转导</td><td> 2348L </td><td></td></tr><tr><td>抗-Chk1的抗体</td><td>圣克鲁斯</td><td> SC7898 </td><td></td></tr><tr><td>抑肽酶</td><td> MP Biomedicals </td><td> 0219115880 </td><td></td></tr><tr><td>放线菌酮</td><td>西格玛</td><td> C7698-5G </td><td></td></tr><tr><td>细胞松弛素B </td><td> EMD </td><td> 250233 </td><td></td></tr><tr><td>二硫苏糖醇（DTT）的</td><td> VWR </td><td> JTF780-2 </td><td></td></tr><tr><td>人绒毛膜促性腺激素</td><td>西格玛</td><td> CG10-10VL </td><td></td></tr><tr><td> L-半胱氨酸</td><td>西格玛</td><td> C7352-1KG </td><td></td></tr><tr><td>亮肽素</td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td>甲基磺酸甲酯（MMS） </td><td>西格玛</td><td> 129925-5G </td><td></td></tr><tr><td>诺考达唑</td><td>西格玛</td><td> M1404-2MG </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem公司</td><td> 367222 </td><td></td></tr><tr><td>上样缓冲液</td><td>西格玛</td><td> S3401 </td><td></td></tr><tr><td>互变</td><td>光化工（美国） </td><td> 209-12041 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>产蛋桶</td><td> Rubbermaid的商业产品</td><td> 6308 </td><td></td></tr><tr><td> CL2 IEC离心机转子的摆动</td><td> Thermo Scientific的</td><td> 004260F </td><td></td></tr><tr><td> HB6摆动转子</td><td> Thermo Scientific的</td><td> 11860 </td><td></td></tr><tr><td> SORVALL RC6加超高速离心机</td><td> Thermo Scientific的</td><td> 46910 </td><td></td></tr><tr><td> UV交联</td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 解决方案 </td></tr><tr><td> 1个MMR </td><td></td><td></td><td> 100mM的NaCl，2mM的氯化钾，0.5mM的MgSO 4干燥 ，2.5mM的CaCl 2的 ，5mM的HEPES，pH值调节至7.8，用10 M NaOH </td></tr><tr><td>抑肽酶/亮肽素股票</td><td></td><td></td><td> 10毫克/毫升在水中。将20μL分装-80°C </td></tr><tr><td> x的缓冲区</td><td></td><td></td><td> 15mM的NaCl的80 mM KCl中，0.2M蔗糖，5毫摩尔MgCl 2，1mM的EDTA，10mM的HEPES，pH值调节至7.5，用HCl </td></tr><tr><td>放线菌酮股票</td><td></td><td></td><td> 10毫克/毫升在水中。存储1毫升的等分试样在-20℃。 </td></tr><tr><td>细胞松弛素B股</td><td></td><td></td><td> 5毫克/毫升在DMSO中。 20μL分装-20℃保存</td></tr><tr><td>二硫苏糖醇（DTT）的股票</td><td></td><td></td><td> 1 M中水。存储1毫升的等分试样在-20℃。 </td></tr><tr><td> ELB </td><td></td><td></td><td> 0.25M蔗糖，1mM的DTT，50微克/毫升放线菌酮，2.5毫摩尔MgCl 2，50 mM KCl中，10mM的HEPES，pH7.7 </td></tr><tr><td>诺考达唑股票</td><td></td><td></td><td>在DMSO中的10毫克/毫升。将5微升等分在-80°C </td></tr><tr><td>能量合剂</td><td></td><td></td><td> 375 mM的磷酸肌酸，50mM的ATP，和25mM的MgCl 2。等分试样在-80℃下保存</td></tr><tr><td>核染料溶液</td><td></td><td></td><td> 0.4μg/ ml的Hoechst 33258染色，25％甘油（体积/体积），在1x PBS </td></tr><tr><td>变构股票</td><td></td><td></td><td>在DMSO中的100μM的。在-80℃下存储10微升等份</td></tr></tbody></table>

study of the dna damage checkpoint using xenopus egg extracts

在每天的基础上，将细胞进行的各种内源性和环境的侮辱。为了解决这些侮辱，细胞进化作为一个监督机制，检测DNA损伤和直接的细胞反应对DNA损伤的DNA损伤检验点的信号。有几组的蛋白质称为DNA损伤检查点的信号（ 图1）中所涉及的传感器，传感器和效应器。在这种复杂的信号转导通路，ATR（ATM和RAD3相关）是主要的激酶，可以响应DNA损伤和复制压力之一。激活ATR磷酸化其下游底物，如检查点激酶1（Chk1的）。因此，磷酸化和激活Chk1的导致许多下游的影响，在DNA损伤检验点包括细胞周期阻滞，转录激活，DNA损伤修复，细胞凋亡或衰老（ 图1）。当DNA被破坏，无法激活DNA损伤检验点在UNREP结果播出损伤，且随后，基因组不稳定。细胞DNA损伤检验点的研究阐明了如何保持基因组的完整性，并提供了一​​个更好地了解人类疾病，如癌症，如何发展。 非洲爪蟾卵提取物正在成为一个强大的无细胞提取DNA损伤检验点的研究模型系统，。低速提取物（LSE）最初是由升井组1。去膜精子染色质核形成DNA的半保留复制的方式一次细胞周期LSE结果。 ATR/Chk1-mediated检查点的信号转导通路所引发的DNA损伤或复制压力2。目前使用的两种方法诱导的DNA损伤检查点:DNA损伤的方法和DNA损伤模仿结构体3。 DNA损伤可诱导的紫外线（UV）照射，γ-照射，甲基甲烷esulfonate（MMS），丝裂霉素C（MMC），4 -硝基喹啉-1 -氧化物（4-NQO），或aphidicolin的3，4。 MMS是一种烷化剂，抑制DNA的复制和激活ATR/Chk1-mediated DNA损伤检验点4-7。紫外线照射触发的ATR/Chk1-dependent的的DNA损伤检验点8。的DNA损伤模仿结构AT70退火是一个复杂的两个寡核苷酸的聚（dA的）70和聚-（dT）的70。 AT70比尔·邓菲的实验室系统的开发和广泛使用9-12，诱导ATR/Chk1检查点的信号。 在这里，我们描述协议（1）准备细胞的卵提取物（LSE），（2）治疗爪蟾精子染色质与两种不同的DNA损伤的方法（MMS和UV），（3）准备的DNA损伤模仿的结构AT70，和（4）来触发的ATR/Chk1-mediated DNA损伤检查点LSE相损坏的精子染色质或DNA损伤模仿结构。

Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

非洲爪蟾卵提取物是一个有用的模型系统，研究DNA损伤检验点。该协议是非洲爪蟾卵提取物和DNA损伤检验点的诱导试剂的准备。这些技术是适应各种DNA损伤的方法，在研究的DNA损伤检查点的信号。

研究DNA损伤检验点<em&gt;爪蟾</em&gt;卵提取物

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

study-of-the-dna-damage-checkpoint-using-xenopus-egg-extracts

Research

JoVE Journal

Biology

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

16.6K 次观看.  University of North Carolina at Charlotte. 非洲爪蟾卵提取物是一个有用的模型系统，研究DNA损伤检验点。该协议是非洲爪蟾卵提取物和DNA损伤检验点的诱导试剂的准备。这些技术是适应各种DNA损伤的方法，在研究的DNA损伤检查点的信号。

视频: Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

重组β-catenin的降解<em&gt;爪蟾</em&gt;卵提取物

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

研究DNA损伤检验点

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