通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

摘要

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

摘要

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

引言

The two most reliable methods of identifying radiation-induced inter-chromosomal stable aberrations are multiple fluorescence in situ hybridization (mFISH), which allows the painting of two or more chromosomes simultaneously, and spectral karyotyping (SKY), which imparts a distinct color to each homologous chromosome pair in the genome. Unlike unstable aberrations, stable aberrations are persistent in nature and may be propagated for several generations in irradiated populations¹, and are regarded as critical molecular "signatures" of radiation-induced cytogenetic lesions². Studies by various groups have shown that stable aberrations are associated with the pathogenesis and development of a number of diseases, including cancer³. Before the era of chromosome painting (also referred as molecular cytogenetics), the conventional G-banding technique was the only method for detecting stable chromosomal aberrations. However, chromosome banding is a challenge to cytogeneticists because the resolution is limited, reproducibility is uncertain, it is a labor-intensive procedure, and it requires highly skilled and experienced cytogeneticists for reliable data interpretation⁴. Moreover, the classic banding technique does not allow detection of complex chromosomal rearrangements, which involve the interaction of three or more breaks distributed among two or more chromosomes, a common outcome of radiation damage. Complex aberrations may persist in individuals many years after radiation exposure, making them useful for retrospective biodosimetry⁵. Therefore, an alternate approach was required to overcome the limitations of conventional banding techniques to detect stable chromosomal rearrangements.

In the late 1960s, the pioneering work of Gall and Pardue (1969) on molecular hybridization using nucleic acid probes labeled with radioactive material commenced a new era in the field of cytogenetics, which allowed detection of a specific DNA sequence on chromosomes⁶. However, the use of radioactive probes for molecular hybridization had several drawbacks: radioactive probes are relatively unstable, probe activity depends on radioactive decay of the isotope used, hybridization takes a longer time, the resolution is limited, the probes are relatively costly, and the radioactive materials are a health hazardous. Thus, it became necessary to develop and design non-radioactive probes. The introduction of fluorescent tagged nucleic acid probes in the 1980s and 1990s overcame the limitations of radioactive probes and greatly enhanced the safety, sensitivity, and specificity of the hybridization technique^7-10. Fluorescent probes give rise to extremely bright signals when observed under fluorescence microscopes equipped with the appropriate excitation and emission filters. Any loss, gain, or rearrangement of fluorescent labeled chromosome/s or a part of the chromosome is easily identifiable with this FISH technique.

Analysis of chromosomal aberrations by FISH painting has led to marked progress in cytogenetic research over the years. Designing fluorescent labeled probes for specific applications ranging from locus-specific probes to whole-chromosome painting probes has advanced the field significantly; this has also facilitated the detection of submicroscopic ("cryptic") rearrangement, which was not possible by conventional chromosome banding. Chromosome painting by mFISH and SKY have proven to be valuable tools for the identification of simple and complex inter-chromosomal rearrangements. The basic principles for both techniques are similar, but the method of detection and discrimination of fluorescent signal after in situ hybridization and the process of image acquisition are different. In mFISH, separate images of each of the four fluorochromes are captured by using narrow bandpass microscope filters; dedicated software is then used to combine the images. While in SKY, image acquirement is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy, which allows the measurement of the entire emission spectrum with a single exposure at all image points. In both mFISH and SKY, monochrome images are captured independently, then merged, and finally, unique pseudo-colors are assigned to the chromosomes in monochromatic images based on the specific dye attached to each fluorochrome probe.

The contribution of mFISH and SKY analysis in the radiation biology field is remarkable, particularly for retrospective dose estimation of human exposure to IR (radiation biodosimetry)^11-14, radiation carcinogenesis risk assessment¹⁵, as well as detection and risk estimation of radiotherapy-related secondary cancer¹⁶. A recent study on mice has shown that a FISH-based chromosome painting technique is also an important tool for evaluating the efficacy of radiation countermeasure¹⁷. In the present study, the effect of total body radiation exposure on the induction of stable chromosomal aberrations in the bone marrow cells of mice has been demonstrated using mFISH and SKY techniques.

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研究方案

所有的动物研究都严格按照该指南中的建议，美国国立卫生研究院的实验动物的护理和使用进行。动物方案经阿肯色大学医学科学机构的动物护理和使用委员会。新鲜空气和向标准啮齿动物食物和自由饮水15小时周期 - 所有动物在20±2℃，10在标准空调动物设施收纳。抵达后，小鼠隔离举行1周，并提供认证的啮齿动物食物。

1.Radiation曝光并在中期细胞的体内逮捕

1. 揭露联合国麻醉小鼠2戈瑞全身辐射照射室。在辐照过程中，在通风良好的固定室老鼠的地方，以确保他们不会自由移动，并获得一致的剂量。
2. 注入100微升0.05％秋水仙素溶液（c溶解的钙和镁的PBS olchicine粉末）腹膜内使用连接用1mL一次性注射器将25g针。避免直接注射到任何器官。
3. 留在笼子里动物的骨髓细胞收获前2小时。观察疼痛或痛苦的任何迹象的动物。

2.骨髓细胞采收和密度梯度离心骨髓单个核细胞的分离

1. 安乐死的CO ₂窒息鼠标。
2. 喷在背腹侧70％的乙醇。
3. 就用锋利的剪刀腹部皮肤2厘米的切口。抓住钝镊子切口两侧皮肤，轻轻拉开腹部肌肉。
4. 切下的后腿用剪刀，并立即将它们放置在预冷的PBS，4％FBS。
5. 仔细清理附着于后肢用锋利的剃刀刃和棉花纱布包扎的所有肌肉。
6. 分离从股骨胫骨。修剪用剃刀刃每个骨骼的两个技巧。
7. 冲洗骨髓用3mL的PBS（含4％的FBS）的使用连接到一个3毫升注射器23政针的内容，并收集在15毫升离心管中的内容。通过细胞悬浮液的至少10倍通过针，使单细胞悬浮液。
8. 小心覆盖等体积的淋巴细胞分离介质（3mL）中对细胞悬浮液，而不会干扰细胞悬浮液和淋巴细胞分离介质之间的接口。离心在400 xg离心在室温下30分钟。
9. 小心收集血沉棕黄层，而不会干扰其它层和传输在新的15毫升离心管中。

3.中期细胞价差的制备

1. 加入10 mL的PBS含有血沉棕黄层的管。
2. 离心在400 xg离心在室温下5分钟。
3. 小心取出上清液。然后分手Ť他细胞沉淀，轻轻敲击，并添加10毫升的PBS。
4. 离心在400 xg离心在室温下5分钟。
5. 而不干扰细胞团块除去上清，打散沉淀，并添加4mL的预热低渗0.075 1M氢氯化物溶液。通过添加温和持续振荡下跌低渗溶液滴。
6. 孵育细胞20分钟，在37℃的水浴中。
7. 低渗处理后，添加固定剂的等体积（4mL）中（甲醇:冰醋酸，3:1体积/体积）中的15毫升管中并通过反相管轻轻混匀。
8. 离心在400 xg离心在室温下5分钟并弃上清。
9. 分手的细胞沉淀用攻丝接着温和涡流几秒钟，并通过与恒定振荡滴加入3毫升固定剂溶液滴。
10. 孵育在室温下30分钟，然后在400 xg离心离心5分钟。
11. 除去上清液，分手绅士细胞沉淀乐攻，加3毫升新鲜固定液。
12. 离心在400 xg离心在室温下5分钟，并除去上清液。分手轻轻敲击细胞沉淀，并加入新鲜的3毫升固定液。
13. 重复步骤3.12两倍多。
14. 离心在400 xg离心在室温下5分钟，并除去上清液而不扰乱细胞沉淀。然后加入400〜600μL固定液和吹打调匀。
15. 滴30微升固定的细胞上以45°角倾斜的预清洗和湿载玻片并允许滑动到空气完全干燥过夜。

通过渔业部4小鼠染色体绘画

1. 染色体变性
   1. 将包含中期细胞利差2X SSC在室温下2分钟的幻灯片。
   2. 脱水串行乙醇洗涤滑板为70％每2分钟，80％，和100％。在65℃下孵育60分钟。
   3. 预热40毫升变性溶液（70％甲酰胺/ 2×SSC; pH值7.0）至70℃（±2℃）下在玻璃科普林缸30分钟。 1.5分钟 - 通过温育在预热的变性溶液滑动1变性染色体。
   4. 骤立即滑到在冰冷的70％乙醇2分钟，停止变性过程以及防止变性染色体从重新退火。然后，通过用80％乙醇洗涤2分钟脱水。重复用100％乙醇的乙醇洗涤2分钟。完全干燥后在室温下的幻灯片。
2. 变性和探针混合物杂交
   1. 短暂离心由制造商提供的探针混合物，在80℃（±2℃）的水浴中传送探测混合液10μL到500μL的卡扣帽离心管中，和变性温育7分钟。
   2. 将带有探针混合物的离心管在水浴中于37℃10分钟。
   3. 变性探针混合物应用到与变性chromoso幻灯片MES。
   4. 仔细覆盖18毫米×18毫米的玻璃盖玻片的区域以及非常轻轻按压盖玻片滑动消除任何可见的气泡。密封用橡胶水泥盖玻片的所有四个侧面和在用于杂交的潮湿室中在37℃孵育12小时，在黑暗中16小时。
3. 杂交后清洗和检测
   1. 小心取出橡胶水泥和盖玻片。
   2. 在74℃（±2℃）下5分钟在预温0.4X SSC地方滑动。
   3. 在2分钟的洗涤溶液II（4×SSC / 0.1％吐温-20）洗涤滑动。
   4. 放置用DAPI复染（1.5微克/毫升）的抗褪色封固剂的20微升，用玻璃盖玻片覆盖。轻轻按下与实验室组织盖玻片，以去除气泡和多余的安装解决方案。用指甲油密封盖玻片的边缘。
   5. 使用配备有适当的过滤器的荧光显微镜视图幻灯片。

5.光谱核型分析小鼠染色体的（SKY）

1. 染色体和探针变性
   1. 平衡滑动在2×SSC在室温下进行2分钟，不摇动。
   2. 脱水在乙醇系列（100％乙醇70％，80％，和），在室温下每2分钟幻灯片。空气干燥幻灯片完全去除乙醇。
   3. 在玻璃科普林缸30分钟暖40毫升变性溶液（pH 7.0 70％甲酰胺/ 2×SSC）。
   4. 孵育在1至1.5分钟预热的变性溶液滑动。
   5. 立即浸入滑动入冰冷的70％乙醇2分钟，停止变性过程以及防止变性染色体从重新退火。
   6. 通过在室温下将其放入80％乙醇中2分钟，然后为100％乙醇2分钟，脱水的幻灯片。
   7. 在设定为80℃（±2℃）水浴变性SKY探头（瓶＃1由制造商提供）7分钟，然后立即放入设定为37℃，10分钟的不同水浴中。
2. 探针杂交
   1. 添加变性SKY探针的10微升到变性染色体。
   2. 小心地将18毫米×18毫米的玻璃盖玻片冲上天空探头，以便没有气泡盖玻片被困。
   3. 密封用橡胶水泥盖玻片的边缘。
   4. 放置幻灯片在加湿遮光室并在37℃下在黑暗中孵育24小时至36小时。
3. 杂交后的洗涤和荧光检测
   1. 不干扰盖玻片非常小心取出橡皮泥。
   2. 放置滑入预热快速洗涤液（0.4倍SSC）在72℃（±2℃）下5分钟，以恒定振荡。沉浸滑入洗涤溶液III（4×SSC / 0.1％吐温20）和孵育1分钟，同时振摇。
   3. 可选:加入80微升阻断剂的（小瓶＃2由制造商提供的）上的杂交的区域中，放置一个24毫米×60毫米的塑料盖玻片，并在37℃下在黑暗中温育30分钟，在湿润的腔室中。
   4. 轻轻取出塑料盖玻片和洗涤用预热（45℃）洗涤溶液III滑动5分钟。
   5. 应用80 Cy5信号μL染色试剂（瓶＃3制造商提供），放置24毫米×60毫米的塑料盖玻片，在黑暗中，在加湿室内37℃下孵育40分钟。
   6. 浸滑入含有玻璃科普林缸预热（45℃）洗涤溶液III（4×SSC / 0.1％吐温20），并在水浴中于45℃孵育2分钟，振摇。重复此洗涤步骤3次。
   7. 应用80经Cy5.5的μL染色试剂（瓶＃4由制造商提供），放置24毫米×60毫米的塑料盖玻片，在黑暗中，在加湿室内37℃下孵育40分钟。
   8. 与预热洗幻灯片3次（45℃）洗涤液III。
   9. 持反对纸巾倾斜位置排出多余液体的幻灯片。添加20μL的抗褪色DAPI试剂（小瓶＃5由制造商提供），并小心地将24毫米×60毫米的玻璃盖玻片而不引入任何气泡。密封盖玻片的边缘与指甲油和与配备用于捕获SKY图像的落射荧光显微镜观察。

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结果

全身照射诱导照射小鼠的骨髓细胞大量染色体畸变。当前协议是用于治疗辐射暴露后在体内的骨髓细胞的有丝分裂停滞优化，从照射小鼠，骨髓单核细胞的分离的后腿骨髓细胞的收获通过密度梯度离心，制备中期细胞价差，以及随后的检测由渔业部和SKY技术辐射诱导稳定染色体畸变。染色体画使用这些技术可用于检测和评估各种病理生理条件的危险。

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讨论

几个关键步骤，确定渔业部和天空的成功。第一个也是最关键的一步是优化体内骨髓单个核细胞有丝分裂逮捕秋水仙碱治疗。秋水仙素浓度和处理时间是单独或共同确定有效的染色体绘画有丝分裂指数以及染色体浓缩，两个重要的先决条件。高浓度的秋水仙素或更长的治疗时间导致高度浓缩的染色体，不兼容的适当变性和杂交。此外，与冷凝染色体流传中期细胞复合重排的准确识别并不是?...

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材料

Name	Company	Catalog Number	Comments
Formamide	Sigma-Aldrich	221198-100ML
SSC Buffer 20× Concentrate	Sigma-Aldrich	S6639-1L
SKY Laboratory Reagent for Mouse	Applied Spectral Imaging	FPRPR0030/M40
CAD - Concentrated Antibody Detection Kit	Applied Spectral Imaging	FPRPR0033
Single Paints Customized - 3 Colors; Mouse chromosome 1: Red, Mouse chromosome 2: Green, Mouse chromosome 3: Aqua	Applied Spectral Imaging	FPRPR0182/10
Glass coverslips	Fisher Scientific	12-545B
Tween 20	Fisher Scientific	BP337-100
Hydrochloric acid, 37%, Acros Organics	Fisher Scientific	AC45055-0025
Fisherbrand Glass Staining Dishes  with Screw Cap	Fisher Scientific	08-816
KaryoMAX Potassium Chloride Solution	Life Technologies	10575-090
Fisherbrand Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Colcemid powder	Fisher Scientific	50-464-757
Histopaque-1083	Sigma-Aldrich	10831
Shepherd Mark I, model 25 137Cs irradiator	J. L. Shepherd & Associates	Model 484B
Syringe 1 mL	BD Biosciences	647911
Ethyl Alcohol, 200 Proof	Fisher Scientific	MEX02761
PBS, (1x PBS Liq.), w/o Calcium and Magnesium	Fisher Scientific	ICN1860454
Fetal Bovine Serum	Fisher Scientific	10-437-010
Methanol	Fisher Scientific	A454SK-4
Glacial acetic acid	Fisher Scientific	AC295320010
Zeiss Microscope	Zeiss	AXIO Imager.Z2