Name: measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation
Uploaded: 2015-07-03T17:00:00
Description: Watch this Scientific Journal Video about Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation at JoVE.com

The authors would like to acknowledge the contribution of our colleagues Sandrine Roch-Lefevre and Eric Gregoire of the Institute of Radioprotection and Nuclear Safety (Paris, France). This work was supported by the Government of Canada Science and Technology program at Canadian Nuclear Laboratories (Chalk River, Ontario, Canada), the CANDU Owners Group (Toronto, Ontario, Canada), the Canadian Nuclear Safety Commission and by the Institute of Radioprotection and Nuclear Safety (Paris, France).

所有的鼠标操作和处理程序应遵循规定由立法机关和/或动物保健计划，并批准由当地动物保护委员会的规则。在这个协议中描述的所有方法均按照加拿大理事会关于动物保护与当地动物保护委员会批准的指导进行。 注意：有放射性的所有工作（包括但不限于处理氚，γ外照射，放射性处理的动物组织，床上用品废物）要坚持规定由立法机关和/或辐射防护部门的规则，并授权执行人员经过认证​​的实验室?...

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S., Girard, C., Klokov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA+double-strand+breaks+is+not+modulated+by+low-dose+gamma+radiation+in+C57BL/6J+mice.">Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.</a> Radiat Res. 1815 (5), 548-559 (2014).</li><li>Runge, R., 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=Fully+automated+interpretation+of+ionizing+radiation-induced+gammaH2AX+foci+by+the+novel+pattern+recognition+system+AKLIDES(R).">Fully automated interpretation of ionizing radiation-induced gammaH2AX foci by the novel pattern recognition system AKLIDES(R).</a> Int J Radiat Biol. 88 (5), 439-447 (2012).</li><li>Jucha, 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=FociCounter:+A+freely+available+PC+programme+for+quantitative+and+qualitative+analysis+of+gamma-H2AX+foci.">FociCounter: A freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.</a> Mutat Res. 696 (1), 16-20 (2010).</li><li>Garty, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RABIT:+a+rapid+automated+biodosimetry+tool+for+radiological+triage.">The RABIT: a rapid automated biodosimetry tool for radiological triage.</a> Health Phys. 98 (2), 209-217 (2010).</li><li>Kovalchuk, I. 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本文提出的协议是用于进行大规模的老鼠体内研究审查低水平各种化学和物理因素，包括电离辐射的遗传毒性作用是有用的。我们独特的无特定病原体动物设施配备了150 GammaBeam辐射和一个30米长的照射大厅允许进行涉及低或非常低剂量率照射在数百甚至数千老鼠终身学习。较小的辐照设施慢性低剂量照射，可设置在一所大学或医院的环境，放射生物学的研究进行例行和辐射防护指导方针/法...

显著争论低或非常低剂量的电离辐射和公众的恐惧辐射，由广岛和长崎原子弹爆炸和珍稀核电站事故的图像驱动的潜在有害影响（通过大众媒体加剧），导致了很严格的辐射防护监管和有潜在不能科学合理的标准。在过去的三十年中，许多报告已经证明了两者的不足和有害诱导低剂量辐射1-4潜在有益的生物效应的存在。主要辐射的健康危险因素是癌症的概率，根据对原子弹爆炸幸存者接受高或中等剂量的辐射流行病学研究估计。这些数据（所谓的线性无阈值或LNT模型）的线性外推来估算的癌症风险在低剂量。然而，这种方法并没有收到世界通用的科学验收并被大量辩论5。 很明显，更多的研究来澄清这一问题，并可能提高辐射防护标准。这些研究应当包括（最好为外推效果对人类）和这样的最终点作为DNA损伤率，DNA修复和诱变慢性治疗（环境和职业暴露最佳逼近）， 在体内动物模型。已知的是，DNA的是用于破坏性辐射效应和不完整或错误修复可导致诱变和癌发展6的主要目标。 DNA双链断裂（DSB）是最有害类型的DNA损伤之一，并且可能会导致细胞死亡和肿瘤发生7。据，因此，重要的是能够可靠地和精确地测量DSB暴露于低剂量辐射和/或其他压力，如化学污染物后的水平。其中最敏感和特异的标记物的DNA DSB的是磷酸化组蛋白H2AX，称为γH2AX8，但其他标记物和方法已被建议9,10。据估计，数千H2AX分子，在诱导的DSB的附近，是参与γH2AX的形成可进行个体的DSB的通过免疫荧光标记的检测与抗γH2AX抗体和荧光显微镜11。该反应是非常快的，分30至60达到其最大值。有证据表明γH2AX利于修复DNA DSB通过吸引其他修复因子来休息的场所，并通过改变染色质结构来断锚定DNA末端，并提供访问其他修复蛋白（12综述）。在修复DNA DSB的完成，得到γH2AX去磷酸化和/或发生退化，新合成的分子H2AXγH2AX替代染色质10的受灾地区。监测γH2AX的形成和损失，因此，提供的DNA DSB修复动力学的精确估计。这种方法已被用于研究修复DSB在照射高剂量的辐射和其速率和残留的DSB水平的各种人肿瘤细胞系已被证实与放射敏感性13-15。 我们修改了这个实验方法并将其应用到小鼠体内研究，探讨低剂量慢性γ-和β辐射对DNA DSB水平和维修（图1）的影响。首先，我们展示了一个方法来执行的小鼠的长期慢性暴露于β辐射由氚或者发射（氢-3）中氚化水（HTO）的形式或作为有机结合的氚（OBT）溶解在饮用水。这两种形式都预期累积和/或分发不同的机构，因此，产生不同的生物学效应。这两种形式是核工业的潜在危险。这种治疗方法由长期暴露于γ射线在等效剂量率并联以允许正确比较的两个辐射类型，这对于它们的相对生物学有效性的评价是至关重要的。的β-辐射是由电子的，使得它从γ射线，高能光子非常不同。由于这种差异，Β辐射表示主要是内部的健康危害和可能产生较γ射线不同的生物学效应。这种并发症导致曝光过度的监管，β射线通过HTO发出显著争议。因此，HTO在饮用水的公共管理水平各不相同，从100贝可/ L的欧洲至75,000贝可/ L，在澳大利亚。它是，因此，重要的是要比较HTO生物效应等效剂量γ射线的。其次，DNA DSB的速率是使用免疫荧光标记的γH2AX检测完成慢性暴露后测定分离的脾细胞通过流式细胞仪主编。这允许通过在体内暴露造成DNA损伤的程度的评价。然而，这是明智的预期，这些低水平暴露可能不会产生DNA DSB任何检出率;相反，一些隐藏更改/应答可以预期这将影响到细胞的修复DNA损伤的能力。这些变化中，如果发现，可以是刺激（产生有益的影响）或抑制（产生有害的影响）。该协议允许通过用高剂量的辐射，产生一个显著量损坏的挑战所提取的脾细胞揭示这样的变化（ 例如 ，2戈瑞产生每细胞约50的DNA DSB或3 - 5倍，增加总的γH2AX水平） 。接着，形成与γH2AX的损失，这反映了DSB修复的起始和结束时，通过流式细胞术监测。以这种方式，不仅基底和DNA DSB的治疗引起的水平可以被测量，但还对细胞的能力的潜在影响作出回​​应和修复DNA损伤诱导的高得多的水平应激。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HTO: tritiated water, [3H]</td>
			<td>locally obtained from a nuclear reactor&#160;</td>
			<td></td>
			<td>stock activity 3.7 GBq/mL;&#160; can be substituted with HTO from Perkin Elmer</td>
		</tr>
		<tr>
			<td>OBT: Alanine, L-[3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET348005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Glycine, [2-3H]: organically bound tritium</td>
			<td>Perkin Elmer</td>
			<td>NET004005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Proline, L-[2,3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET323005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>tritiated water, [3H] (HTO)</td>
			<td>Perkin Elmer</td>
			<td>NET001B005MC</td>
			<td>substitute for HTO of local origin&#160;&#160;</td>
		</tr>
		<tr>
			<td>GammaBeam 150 irradiator</td>
			<td>Atomic Energy of Canada Limited</td>
			<td>locally manufactured</td>
			<td>can be substituted with another g-radiation source of sufficiently low activity&#160;</td>
		</tr>
		<tr>
			<td>tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Fisher Scientific</td>
			<td>SH3025501</td>
			<td>Hyclone RPMI 1640 with L-Glutamine and HEPES 500mL</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Sigma Aldrich</td>
			<td>F1051-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-gH2AX antibody, clone JBW301</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor-488 goat anti-mouse antibody</td>
			<td>Life Technologies (formerly Invitrogen)</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodine</td>
			<td>Sigma Aldrich</td>
			<td>P4864-10ML</td>
			<td>1mg/mL</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P006-EAAN</td>
			<td>500ml bottles Absolute Ethanol</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>2682550</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td>sterile polypropylene centrifuge tubes</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Linde</td>
			<td>P110403</td>
			<td></td>
		</tr>
		<tr>
			<td>12 x 75 mm mL uncapped glass tubes</td>
			<td>Fisher Scientific</td>
			<td>K60B1496126</td>
			<td>disposable borosilicate glass tubes with plain end</td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>VWR</td>
			<td>CA15708-120</td>
			<td>nunc tisue culture 25ml flask (supplier no. 156340)</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
			<td>Wagner scissors 12cm sharp/sharp</td>
		</tr>
		<tr>
			<td>forceps, straight</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td>Semken forceps 13cm, straight</td>
		</tr>
		<tr>
			<td>forceps, curved</td>
			<td>Fine Science Tools</td>
			<td>11003-12</td>
			<td>Narrow Pattern forceps 12 cm, curved</td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td>VWR</td>
			<td>CA25382-100</td>
			<td>BD Falcon tissue culture dish 60x15mm</td>
		</tr>
		<tr>
			<td>cell strainers, 70 mm</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td>Falcon cell strainers 50/case</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS recipe: 1 tablet dissolved in 200mL of deionized water, adjust pH to 7.4 if needed.</td>
			<td>Sigma Aldrich</td>
			<td>P4417-100TAB</td>
			<td>Phosphate Buffered Saline Tablets</td>
		</tr>
		<tr>
			<td>TBS recipe: to make 10X Stock,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30g TRIS HCl</td>
			<td>Sigma Aldrich</td>
			<td>T3253-1KG</td>
			<td>Trizma hydrochloride</td>
		</tr>
		<tr>
			<td>88g NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Sodium Chloride</td>
		</tr>
		<tr>
			<td>2g KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Potassium Chloride</td>
		</tr>
		<tr>
			<td>Dissolve in 1L of deionized water, adjust pH to 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation

低剂量的辐射暴露可能产生多种生物效应是在由高辐射剂量所产生的影响的数量和质量不同。处理适当和有科学依据的方式与环境，职业健康和公共卫生安全问题在很大程度上依赖于准确测量低剂量的污染物，如电离辐射，化学物质的生物效应的能力。 DNA损伤和修复是健康风险最重要的早期指标，由于其潜在的长期后果，例如癌症。在这里，我们描述了一种协议来研究慢性体内暴露于低剂量的γ-和β射线对DNA损伤和修复在小鼠脾细胞中的作用。采用DNA双链断裂的一个普遍接受的标记，磷酸化组蛋白H2AX被称为γH2AX，我们展示它如何被用于评估DNA损伤的不仅是水平，而且还改变在DNA修复能力可能通过体内暴露低剂量制备。流式细胞仪允许在大量样品的免疫荧光标记的γH2AX的快速，准确和可靠的测量。 DNA双链断裂修复可以通过暴露提取脾细胞的2戈瑞一个具有挑战性的剂量以产生足够数量的DNA的破坏触发修理和通过测量诱导（1小时后照射）和残留的DNA损伤进行评估（24小时照射后）。残余DNA损伤将指示不完全修复和长期的基因组不稳定和癌症的风险。结合其它测定法和最终点，可以很容易地在这种被测定体内研究（ 例如 ，染色体畸变，在骨髓的网织红细胞微核频率，基因表达等），这种方法允许的生物效应的准确和上下文评价低水平压力。

图2示出流的例子术预期脾图形使用此处描述的方法制备。根据&lt;电子音量侧散射&gt;散点图细胞被第一选通（ 图2A和2D;电子体积相当于前向散射）。 FL3 /碘化丙直方图（ 图2B和2E）确认正常细胞周期分布。意味着使用FL1通道计算γH2AX信号证实了&gt; 2倍的增加的DNA DSB在2-戈瑞照射细胞（ 图2F）相比于未处理的对照（ ?...

Watch this Scientific Journal Video about Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation at JoVE.com

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Research

JoVE Journal

Biology

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

一个高度可重复的和直接的方法来执行<em&gt;体内</em&gt;眼剜除术中的鼠标眼睛开放后

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

Measuring Glutathione-induced Feeding Response in Hydra

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

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

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

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 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...