结合DNA-RNA荧光<em>原位</em>杂交（FISH）研究X染色体失活的分化雌性小鼠胚胎干细胞
Summary
荧光原位杂交（FISH）可在细胞内其天然环境中的核酸的检测。我们在这里描述了一种协议，用于组合，同时检测RNA和DNA的鱼的装置，它可以被用来研究X染色体失活在小鼠胚胎干细胞。
Abstract
荧光原位杂交（FISH）是一种分子技术，它可以在细胞中的核酸的检测。 DNA FISH通常用于细胞遗传学和癌症的诊断，而且可以检测的基因组中，它通常具有重要的临床意义的像差。核糖核酸FISH技术可被用于检测RNA分子在细胞内，并提供了在基因表达调控的重要见解。在同一小区内结合DNA和RNA的鱼在技术上具有挑战性，因为适用于DNA FISH条件可能过于苛刻脆弱的，单链RNA分子。我们在这里提出了一个易于应用协议，它使结合起来，同时检测的Xist RNA和DNA的由X染色体编码。这个结合DNA-RNA FISH协议可能可以应用于其他系统，其中RNA和DNA需要被检测到。
Introduction
通过荧光的手段原位杂交（FISH）研究细胞和组织有，自20世纪70年代后期1引言，让研究者可以研究基因，染色质组织和基因表达在亚细胞水平。 DNA FISH经常用于细胞遗传学，染色体组型2，癌症诊断3和植入前遗传筛查4，并具有在分子研究5-6中起重要作用，因为它允许在其天然环境中的核酸的检测。通过RNA鱼单细胞表达分析可以检测本地初级转录和非编码RNA的染色体被转录，并提供优势的评估在群体水平上的基因表达的其他技术，例如包括定量RT-PCR，全基因组表达分析或Northern印迹。通过可视化的RNA转录直接从原产他们的网站发起，它已经例如被注意到，基因表达是随机7，和有时是等位基因特异性8。在技术改进甚至允许细胞9-11内单个mRNA分子的检测和定量。
FISH的基本原理包括的小区内的核酸杂交到由高度特异性Watson和Crick碱基配对的装置的核酸探针。该探针可以是直接或间接地检测到，从而导致可在显微镜可视化的一个信号。最初的尝试包括放射性标记的探针，基于安全问题，有限的空间分辨率和探测只有一个目标在12-14时的能力，而这是有缺陷的。的放射性标记，其中包括荧光染料，半抗原和酶的后续发展，已经允许广泛使用FISH技术作为一种常规的分子生物学技术。将FISH探针可以被直接标记与fluorochROMES由荧光分子化学连接到核酸序列15和一体化荧光标记的核苷酸16-19，或者所述探针可以结合的半抗原（包括生物素和地高辛），并通过半抗原缀合的特异性抗体的免疫学检测半抗原后可以间接地可视化的荧光报告分子20-21。后一种方法允许信号放大通过使用用于增强原始信号的荧光标记抗体的若干层，并且使其中被表达在低水平RNA种类的检测。通过结合直接和间接标记技术和各种半抗原，多个目标，可以同时在同一小区内的可视化。
其中一个在鱼的协议中最重要的步骤之一是探测到目标的杂交。虽然在理论上，一个探测器将仅特异性结合其靶标，在实践中，这种特异性并不总是实现，探针可结合到同源的区域，和杂交条件并不总是理想的目标DNA区域或RNA种类。因此，特别重要的是杂交后洗涤的，因为它们可以增加对FISH程序的严格性，并且可以防止FISH探针，这将导致背景噪声较高水平的非特异性结合。如RNA分子是单链，他们可以很容易地杂交到一个FISH探针。与此相反，在双链DNA分子首先需要的变性步骤，在之后的探针可以杂交。这通常是通过加热的试样，这会导致该DNA的变性来实现。然而，这些苛刻的条件下，易碎，单链RNA分子可能会丢失。因此，结合DNA-RNA FISH需要的条件显著优化，并且更具有技术挑战性相比，只有RNA或DNA的分离检测。
在这里，我们PRESEN结合起来，同时DNA-RNA的鱼让我们来研究X染色体失活（XCI）在鉴别雌性小鼠胚胎干细胞22-24的TA详细的协议。 XCI是一个关键的表观遗传机制，为女性胚胎发育25，结果在heterochromatinization，因此沉默，女性26-27个人的两条X染色体中的一个。必不可少的这个过程是在非编码RNA Xist的 28-30，这是由RNF12 22-23和REX1 24蛋白调节。Xist的表达变得期间胚胎发育中或在体外 ES细胞分化的未来失活的X染色体（Xi）的上调，并且可以沿X染色体分散，从而吸引而导致的X染色体31的转录关机染色质重塑酶。这个扩展的Xist RNA可以通过RNA FISH被形象化为在X染色体上的涂层一些，这也被称为Xist的云。由于雌胚胎干细胞可以失去，由于基因组不稳定的X染色体之一，我们和其他人联合聘请DNA-RNA FISH研究XCI，以确保只有稳定的核型细胞在这一重要进程22,32的分析评估-34。如同每分子生物学技术，几种不同的优良协议已经公布35-38。在这里，我们提出我们的方法，从分化的小鼠胚胎干细胞，细胞固定，FISH探针由尼克 – 翻译，前处理固定细胞的标记的诱导开始允许通透和随后的探头摄取，探头到杂交目标，最后检测探头通过荧光标记的抗体。在本文提出的协议允许的忠实检测的Xist RNA和一个为期两天内的X染色体，而这种技术的基础知识可能可以适应加时赛她的系统和研究领域。
Protocol
Representative Results
Discussion
结合DNA-RNA鱼可以在技术上具有挑战性，因为适用于DNA FISH条件可能过于苛刻不太稳定的RNA分子。几种方法已经被用于研究DNA和RNA在相同的小区，从而无需采用同时孵育探针检测两者的DNA和RNA。例如，在一个叠加方法中，第一RNA FISH被执行，并且细胞进行成像，并且坐标被采取。随后，相同的载片用于DNA鱼，在此期间，核糖核酸FISH的信号丢失。 DNA FISH后，将细胞再成像，并从二者的RNA和DNA的鱼获得?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank all previous and present members of the department of Reproduction and Development of the Erasmus MC for helpful and stimulating discussions during the past years. We also want to thank the three anonymous reviewers for providing helpful feedback. Work in the Gribnau lab is supported by funding from the Dutch research council (NWO-VICI) and an ERC starting grant.
Materials
| female mouse embryonic stem cells | will be made available upon request | ||
| mouse embryonic fibroblasts | can be derived from E13.5 embryos, or via commercial sources e.g Millipore | ||
| ES cell culture medium | DMEM, 15% foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 0.1 mM ß-mercaptoethanol, and 1000 U ml-1 LIF. Filter sterilize and store at 4ºC for maximal 2 weeks. | ||
| LIF | Chemicon | ESG1107 | |
| DMEM | Invitrogen | 11960085 | |
| Fetal Calf Serum | Invitrogen | 10099141 | |
| Penicillin–streptomycin (100×) | Invitrogen | 15140-122 | |
| Non-essential aminoacids | Invitrogen | 11140-050 | |
| ß-mercaptoethanol | Sigma-Aldrich | M7522 | |
| ES cell differentiation medium | IMDM-Glutamax, 15% heat inactivated foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 37.8 μl/l monothioglycerol and 50 μg/ml ascorbic acid | ||
| IMDM-Glutamax | Invitrogen | 31980-030 | |
| Ascorbic acid (50 mg/ml) | Sigma-Aldrich | A4403 | |
| monothioglycerol | Sigma-Aldrich | M6145 | |
| T25 cell culture dishes | Greiner Bio-one | 690160 | |
| 6-well cell culture dishes | Greiner Bio-one | 662160 | |
| cell culture grade PBS | Sigma-Aldrich | D8537 | |
| Trypsin-EDTA 0.25% (v/v) trypsin/0.2% (w/v) EDTA in PBS | Gibco | 25200-056 | |
| falcon tubes | Falcon | 352196 | |
| 24×24 mm coverslips | Menzle | 780882 | |
| gelatin | Sigma-Aldrich | G1890-100G | prepare a 0.2% gelatin/cell culture PBS solution, and autoclave |
| paraformaldehyde | Sigma-Aldrich | P6148 | prepare 4% PFA/PBS solutions, by dissolving PFA in PBS. Stir at 70 ᴼC until completely dissolved and cool down to room temperature. Aliquots can be stored at -20ᴼC |
| absolute 100% ethanol | Sigma-Aldrich | 32221-2.5L | use absolute 100% ethanol and RNAse free water to make 70% and 90% ethanol solutions |
| RNAse free water | Baxter | TKF7114 | |
| sterile filter tips | Rainin Bio Clean | GP-L10F, GP-L200F, RT-1000F | |
| digoxigenin-labeling kit (DIG-Nick translation) | Roche | 11745816910 | |
| biotin-labeling kit (Biotin Nick translation) | Roche | 11745824910 | |
| Sephadex G50 | Sigma-Aldrich | G5080 | |
| 2 ml syringe | BD Plastipak | 300013 | |
| sterilized cotton | |||
| eppendorf tubes | Eppendorf | 30120086 | |
| yeast tRNA 10 mg/ml | Invitrogen | 15401-029 | |
| Salmon Sperm DNA 10 mg/ml | Invitrogen | 15632-011 | |
| mouse cot-1 DNA 1 mg/ml | Invitrogen | 18440-016 | |
| eppendorf bench top microcentrifuge | Eppendorf | Model 5417R | |
| Eppendorf Refrigerated Centrifuge | Eppendorf | Model 5810R | |
| 2M NaAc, pH 5.6 | Sigma-Aldrich | S7670 | |
| 50+ hybridization solution | 50% formamide, 2x SSC, 50 mM phosphate buffer pH 7, 10% dextran sulfate pH 7 | ||
| formamide | Acros organics | 3272350000 | |
| dextran sulfate | Fluka | 31403 | |
| pepsin | Sigma-Aldrich | P6887 | prepare a 0.2% solution in RNAse free water, and add 84 µl 37% HCL per 100 ml |
| HCL, 37% solution | Fluka | 84422 | |
| object glasses | Starfrost | 8037/1 | |
| 20x SSC | Invitrogen | AM9763 | dilute to 2x SSC in RNAse free water |
| 10 mM Phosphate buffer, pH 7 | add 57.7 ml of 1M Na2HPO4 and 42.3 ml 1M NaH2PO4; use DEPC treated water and autoclave | ||
| denaturation buffer | 70% Formamide/2xSSC/10mM phosphate buffer pH 7 | ||
| Tween-20 | Sigma-Aldrich | P2287 | |
| 2M Tris pH 7.5 solution | |||
| Tris-Saline-Tween washing solution (TST) | 100 ml 10x Saline, 50 ml 2M Tris, 500 µl Tween-20, add RNAse free water till 1 liter | ||
| 10x Saline solution | dissolve 85 g NaCl in 1 liter RNAse free H2O, and autoclave | ||
| Tris-Saline-BSA (TSBSA) | 500 μl 10x Saline, 250 μl 2 M Tris, 1 ml 100x BSA, 3,25 ml RNAse free H2O | ||
| BSA, purified, 100x | New England Biolabs | B9001S | |
| sheep-anti-dig antibody | Roche diagnostics | use 1:500 diluted in TSBSA | |
| rabbit-anti-sheep antibody conjugated with FITC | Jackson labs | use 1:250 diluted in TSBSA | |
| goat-anti-rabbit antibody conjugated with FITC | Jackson labs | use 1:250 diluted in TSBSA | |
| mouse-anti-biotin antibody | Roche diagnostics | use 1:200 diluted in TSBSA | |
| donkey-anti-mouse antibody conjugated with Rhodamine | Jackson labs | use 1:250 diluted in TSBSA | |
| goat-anti-horse antibody conjugated with Rhodamine | Jackson labs | use 1:250 diluted in TSBSA | |
| Tris-Saline washing solution (TS) | 10 ml 10x Saline, 5 ml 2 M Tris, 85 ml RNAse free H2O | ||
| Vectashield mounting medium with DAPI | Vectorlabs | H-1200 | |
| nail-varnish | |||
| fluorescent miscroscope |
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