Name: line-1 methylation analysis in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles
Uploaded: 2020-02-01T15:00:00
Description: Watch this Scientific Journal Video about LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles at JoVE.com

这项工作由赫尔辛基大学项目资助（WBS490302，WBS73714112）赫尔辛基大学医院国家资助大学一级健康研究（Y1014SUL05，TYH2016130），芬兰-挪威医学基金会和塞尔玛和马亚-丽莎·塞兰德基金（米内尔瓦基金会）。我们感谢瓦尔特·帕维奇提供经过修改的 MSPA 协议和相关技术支持。我们感谢特穆·马萨林（赫尔辛基大学）帮助我们制作视频。

这项研究得到了赫尔辛基和乌西马医院区道德委员会的批准（道德批准D.217/13/03/02/2015）。
1. 通过超离心制备EV耗尽FBS
<ol><li>将 FBS 放入（超）离心管中，并将其放入超离心桶中。为确保超离心平稳、安全地运行，将水桶彼此平衡在 10 mg 以内。</li>
	<li>在摆动转子上装载铲斗（SW28 型，k 因子 246）。将转子置于超离心机中，在 4°C 下以 100，000 x g运行 19 小时。</li>
	<li>小...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+Cancer:+The+Next+Generation.">Hallmarks of Cancer: The Next Generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Howard, G., Eiges, R., Gaudet, F., Jaenisch, R., Eden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+transposition+of+endogenous+retroviral+elements+in+hypomethylation+induced+tumors+in+mice.">Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice.</a> Oncogene. 27, 404-408 (2008).</li><li>Lander, E. 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P., Ohlsson, R., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epigenetic+progenitor+origin+of+human+cancer.">The epigenetic progenitor origin of human cancer.</a> Nature Reviews Genetics. 7, 21-33 (2006).</li><li>Bock, 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=BiQ+Analyzer:+visualization+and+quality+control+for+DNA+methylation+data+from+bisulfite+sequencing.">BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing.</a> Bioinformatics. 21 (11), 4067-4068 (2005).</li><li>Schouten, J. P., 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=Relative+quantification+of+40+nucleic+acid+sequences+by+multiplex+ligation-dependent+probe+amplification.">Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.</a> Nucleic Acids Research. 30, 57 (2013).</li><li>Nygren, A. O. H., 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=Methylation-Specific+MLPA+(MS-MLPA):+simultaneous+detection+of+CpG+methylation+and+copy+number+changes+of+up+to+40+sequences.">Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.</a> Nucleic Acids Research. 33 (14), 128 (2005).</li><li>El Andaloussi, S., Mager, I., Breakefield, X. O., Wood, M. 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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-+derived+microvesicles:+important+and+underappreciated+mediators+of+cell-to-cell+communication.">Membrane- derived microvesicles: important and underappreciated mediators of cell-to-cell communication.</a> Leukemia. 20, 1487-1495 (2006).</li><li>Lee, T. H., 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=Microvesicles+as+mediators+of+intercellular+communication+in+cancer-+the+emerging+science+of+cellular+"debris".">Microvesicles as mediators of intercellular communication in cancer- the emerging science of cellular "debris".</a> Seminars in Immunopathology. 33, 455-467 (2011).</li><li>Kawamura, Y., Calle, A. S., Yamamoto, Y., Sato, T., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+mediate+the+horizontal+transfer+of+an+active+LINE-1+retrotransposon.">Extracellular vesicles mediate the horizontal transfer of an active LINE-1 retrotransposon.</a> Journal of Extracellular Vesicles. 8, 1643214 (2019).</li><li>Mannerstr&#246;m, B., 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=Epigenetic+alterations+in+mesenchymal+stem+cells+by+osteosarcoma-derived+extracellular+vesicles.">Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles.</a> Epigenetics. 14 (4), 352-364 (2019).</li><li>Shelke, G. V., L&#228;sser, C., Gho, Y. S., L&#246;tvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+exosome+depletion+protocols+to+eliminate+functional+and+RNA-containing+extracellular+vesicles+from+fetal+bovine+serum.">Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum.</a> Journal of Extracellular Vesicles. 3, 24783 (2014).</li><li>Wei, Z., Batagov, A. O., Carter, D. R., Krichevsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+bovine+serum+RNA+interferes+with+the+cell+culture+derived+extracellular+RNA.">Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA.</a> Scientific Reports. 6, 31175 (2016).</li><li>Kornilov, 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=Efficient+ultrafiltration-based+protocol+to+deplete+extracellular+vesicles+from+fetal+bovine+serum.">Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum.</a> Journal of Extracellular Vesicles. 7, 1422674 (2018).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Current Protocols in Cell Biology. 30 (1), 1-29 (2006).</li><li>Puhka, M., 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=Metabolomic+profiling+of+extracellular+vesicles+and+alternative+normalization+methods+reveal+enriched+metabolites+and+strategies+to+study+prostate+cancer-related+changes.">Metabolomic profiling of extracellular vesicles and alternative normalization methods reveal enriched metabolites and strategies to study prostate cancer-related changes.</a> Theranostics. 7 (16), 3824 (2017).</li><li>Peltoniemi, H. H., 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=Stem+cell+enrichment+does+not+warrant+a+higher+graft+survival+in+lipofilling+of+the+breast:+A+prospective+comparative+study.">Stem cell enrichment does not warrant a higher graft survival in lipofilling of the breast: A prospective comparative study.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 66 (11), 1494-1503 (2013).</li><li>Pavicic, W., Joensuu, E. I., Nieminen, T., Peltom&#228;ki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINE-1+hypomethylation+in+familial+and+sporadic+cancer.">LINE-1 hypomethylation in familial and sporadic cancer.</a> Journal of Molecular Medicine. 90, 827-835 (2012).</li><li>. L1.2 sequence on GenBank (accession number: AH005269.2) Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/nuccore/AH005269">https://www.ncbi.nlm.nih.gov/nuccore/AH005269</a> (2000)</li><li>Designing synthetic MLPA probes (v04). MRC-Holland Available from: <a target="_blank" href="https://support.mlpa.com/downloads/files/designing-synthetic-mlpa-probes">https://support.mlpa.com/downloads/files/designing-synthetic-mlpa-probes</a> (2018)</li><li>. Takara Bio Inc Available from: <a target="_blank" href="https://www.takarabio.com/us/products/cell_biology_and_epigenetics/epigenetics/dna_preparation/msre_overview">https://www.takarabio.com/us/products/cell_biology_and_epigenetics/epigenetics/dna_preparation/msre_overview</a> (2018)</li><li>Pisanic, 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=Long+interspersed+nuclear+element+1+retrotransposons+become+deregulated+during+the+development+of+ovarian+cancer+precursor+lesions.">Long interspersed nuclear element 1 retrotransposons become deregulated during the development of ovarian cancer precursor lesions.</a> The American Journal of Pathology. 189 (3), 513-520 (2019).</li></ol>

这项研究说明了如何使用MSPA检测和量化特定遗传元素的甲基化状态。LINE-1是这里的焦点，但探测器可以设计为针对一系列基因和序列。此外，可用于不同应用的探头混合物越来越多。MSPA是一种简单而可靠的DNA甲基化分析技术，不需要双硫酸盐转化10。从样品制备到数据分析的完整过程大约需要 2 天，但仅涉及 4~5 小时的实际动手工作。它适用于少量的DNA（低至70 ng），如此处所?...

DNA甲基化是发生在人类细胞中的一种主要的表观遗传修饰。脱氧核糖核酸甲基化是指甲基组与CpG二核苷酸中细胞氨酸残留物的联系。这种核苷酸通常存在于基因1的5'区域的簇（CpG岛）中。在正常细胞中，大多数的二核苷酸处于非甲基化状态，这允许DNA转录。顺便说一句，许多癌症都与高甲基化CpG岛和转录组沉默2有关，特别是在肿瘤抑制基因中，这反过来又促成了癌症3的各种特征。
另一方面，长穿插的核元素1（LINE-1s或L1s）是重复的、可转位DNA元素，在CpG岛屿通常具有高水平的甲基化。LINE-1的甲基化可防止易位，有助于维持基因组的完整性。在几种类型的癌症中，LINE-1被甲基化，导致活化和随后逆转介导染色体不稳定4。LINE-1占人类基因组5的近17%，其甲基化状态可作为全球基因组甲基化水平6的指标。全球LINE-1低甲基化被认为是在细胞过渡到肿瘤表型7之前;因此，它有望成为早期癌症发病的潜在标志物。
目前，甲基化分析有几种方法，包括热测序、甲基化特异性PCR、微阵列和染色质免疫沉淀1。下一代测序的使用也使得有可能纳入全基因组的DNA甲基化检测方法。这些方法中有许多依赖于双硫酸盐处理的DNA，其中未甲基化的细胞氨酸转化为尿素，甲基化的细胞氨酸保持不变。然而，使用双硫酸盐处理的DNA有几个陷阱，如未甲基化的细胞氨酸不完全转化为尿素，有偏颇的序列扩增，和测序错误8。
在甲基化特异性探针扩增（MSPA）中，由两个寡核苷酸靶向DNA序列的探针靶向DNA序列，该DNA序列含有甲基化敏感限制性酶HhaI9的限制性位点（GCGC）。探针杂交为DNA后，每个样本被分成两组。第一组中的探针进行结扎，而第二组中的探针进行结扎，随后在未甲基化CGCG位点进行HhaI介导消化。两组样品然后由PCR放大，产品通过毛细血管电泳分离。未甲基化位点的探针由HhaI消化，在PCR期间不放大，因此没有峰值信号。相比之下，甲基化位点的探针受到保护，防止消化，因此在PCR期间被放大，随后产生峰值信号10。
与替代方法不同，MSPA 具有几个优点。首先，它需要少量的DNA（50-100 ng），非常适合分析从正式固定石蜡嵌入样品10的DNA。它不需要双硫酸盐处理的DNA;事实上，它不适合以这种方式修饰的DNA。可以同时分析许多样品，并设计MSPA探针，以便它们同时针对多个基因或序列。此外，探针是特定和敏感的甲基化DNA，因为HhaI限制位点对应于一个典型的CpG岛屿10序列。
本研究调查了骨肉瘤（OS）衍生的细胞外囊泡（EV）对脂肪组织衍生的中质性干细胞（AT-MSCs;图 1.EV 是纳米级、膜结合的囊泡，由大多数细胞类型分泌。它们携带蛋白质、脂质、mRNA、微RNA和来自母细胞11、12的其他分子。EV调解细胞间通信，并在几种病理生理条件下发挥重要作用13，14。最近的一项研究显示，癌症衍生的EV可能将活性LINE-1转移到受体细胞15。此前有报道称，除了其他遗传效应16外，来自HOS-143B细胞系的EV可以改变MSCs中LINE-1的甲基化状态。
当为EV分离而生长细胞时，在生长培养基中使用EV耗尽的胎儿牛血清[FBS]很重要，因为FBS衍生的EV可能会干扰来自其他来源的EV，并妨碍结果17，18。超中心化是消耗 FBS EV 的最常见方法之一。与超滤和商业EV耗尽FBS19等替代品相比，这是一个相对简单和具有成本效益的程序。在这里，该协议还演示了如何通过超离心来制备EV耗尽的FBS。
本文为上述技术提供了详细的协议，从从OS细胞系分离EV到在OSEV处理MSC中的LINE-1甲基化分析（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>Terumo</td>
			<td>SS+01T1</td>
			<td>for NTA</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td>MSC cell culture</td>
		</tr>
		<tr>
			<td>3730xl DNA Analyzer</td>
			<td>Applied Biosystems, ThermoFisher Scientific</td>
			<td>3730XL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Optima LE-80K Ultracentrifuge</td>
			<td>Beckman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BlueStar Prestained Protein Marker</td>
			<td>Nippon Genetics</td>
			<td>MWP03</td>
			<td>WB: protein marker</td>
		</tr>
		<tr>
			<td>Calnexin (clone C5C9)</td>
			<td>Cell Signaling Technology</td>
			<td>2679</td>
			<td>WB, dilution 1:800</td>
		</tr>
		<tr>
			<td>CD63 (clone H5C6)</td>
			<td>BD Biosciences</td>
			<td>556019</td>
			<td>WB, dilution 1:1000</td>
		</tr>
		<tr>
			<td>Centrifuge 5702 R</td>
			<td>Eppendorf</td>
			<td>5703000010</td>
			<td>For conditioned media and cells</td>
		</tr>
		<tr>
			<td>Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td>5810000010</td>
			<td>For spinning down 96-well plate</td>
		</tr>
		<tr>
			<td>Centrifuge tube (polyallomer, 14x95 mm)</td>
			<td>Beckman</td>
			<td>331374</td>
			<td>Ultracentrifugation</td>
		</tr>
		<tr>
			<td>DMEM/F-12 + GlutaMAX medium</td>
			<td>Gibco, Life Technologies</td>
			<td>31331-028</td>
			<td>For AT-MSC culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco, Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneScan 500 LIZ size standard</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4322682</td>
			<td>for capillary electrophoresis</td>
		</tr>
		<tr>
			<td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td>
			<td>Sigma</td>
			<td>WGA2-10RXN</td>
			<td>for MSPA negative control</td>
		</tr>
		<tr>
			<td>Hi-Di formamide</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4311320</td>
			<td>for capillary electrophoresis</td>
		</tr>
		<tr>
			<td>HOS-143B cell line</td>
			<td>ATCC</td>
			<td>CRL-8303</td>
			<td></td>
		</tr>
		<tr>
			<td>Hsp70 (clone 5G10)</td>
			<td>BD Biosciences</td>
			<td>554243</td>
			<td>WB, dilution 1:1000</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-mouse</td>
			<td>Li-Cor</td>
			<td>926-32210</td>
			<td>WB: secondary</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-rabbit</td>
			<td>Li-Cor</td>
			<td>926-32211</td>
			<td>WB: secondary</td>
		</tr>
		<tr>
			<td>LINE-1 probe-mix primers</td>
			<td>IDT</td>
			<td></td>
			<td>Sequences in Table 1</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-well reaction plate with barcode</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4306737</td>
			<td>also requires sealing film</td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td>measure protein concentration</td>
		</tr>
		<tr>
			<td>MiniProtean TGX 10% gels</td>
			<td>Bio-Rad</td>
			<td>456-1034</td>
			<td>WB: gel electrophoresis</td>
		</tr>
		<tr>
			<td>NanoSight LM14C</td>
			<td>Malvern Instruments</td>
			<td></td>
			<td>for NTA</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane 0.2 &#181;m</td>
			<td>Bio-Rad</td>
			<td>1620112</td>
			<td>WB: protein transfer</td>
		</tr>
		<tr>
			<td>NucleoSpin Tissue XS</td>
			<td>Macherey-Nagel</td>
			<td>740901.50</td>
			<td>for DNA extraction</td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer</td>
			<td>Li-Cor</td>
			<td>927-40000</td>
			<td>WB: blocking, antibodies</td>
		</tr>
		<tr>
			<td>PBS, 1X</td>
			<td>Corning</td>
			<td>21-040-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco, Life Technologies</td>
			<td>DE17-602E</td>
			<td>Antibiotics for culture media</td>
		</tr>
		<tr>
			<td>Protein LoBind tube, 0.5 mL</td>
			<td>Eppendorf</td>
			<td>22431064</td>
			<td>For storing Evs</td>
		</tr>
		<tr>
			<td>REVERT Total Protein Stain and Wash Solution Kit</td>
			<td>Li-Cor</td>
			<td>926-11015</td>
			<td>WB: total protein staining</td>
		</tr>
		<tr>
			<td>RKO cell line</td>
			<td>ATCC</td>
			<td>CRL-2577</td>
			<td>for MSPA positive control</td>
		</tr>
		<tr>
			<td>RPMI medium 1640 + GlutaMAX</td>
			<td>Gibco, Life Technologies</td>
			<td>61870-010</td>
			<td>For HOS-143B cell culture</td>
		</tr>
		<tr>
			<td>SALSA MLPA HhaI enzyme</td>
			<td>MRC-Holland</td>
			<td>SMR50</td>
			<td></td>
		</tr>
		<tr>
			<td>SALSA MLPA reagent kit</td>
			<td>MRC-Holland</td>
			<td>EK1-FAM</td>
			<td></td>
		</tr>
		<tr>
			<td>SALSA MLPA P300 probe-mix</td>
			<td>MRC-Holland</td>
			<td>P300-100R</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging rotor SW-28</td>
			<td>Beckman Coulter</td>
			<td>342207</td>
			<td>Ultracentrifugation</td>
		</tr>
		<tr>
			<td>Syringe filter, 0.22 &#181;m</td>
			<td>Jet Biofil</td>
			<td>FPE-204-030</td>
			<td>sterile filtering FBS</td>
		</tr>
		<tr>
			<td>Tecnai 12</td>
			<td>FEI Company</td>
			<td></td>
			<td>equipped with Gatan Orius SC 
			1000B CCD-camera 
			(Gatan Inc., USA); for TEM</td>
		</tr>
		<tr>
			<td>TBS, 1X tablets</td>
			<td>Medicago</td>
			<td>09-7500-100</td>
			<td>WB: buffer</td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>WB: transfer</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>ThermoFisher Scientific</td>
			<td>TCA0096</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td>for trypsinization of cells</td>
		</tr>
		<tr>
			<td>TSG101 (clone 4A10)</td>
			<td>Sigma</td>
			<td>SAB2702167</td>
			<td>WB, dilution 1:500</td>
		</tr>
	</tbody>
</table>

line-1 methylation analysis in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles

甲基化特异性探针扩增（MSPA）是一种简单而可靠的技术，可用于检测DNA样本甲基化水平的相对差异。它是足智多谋的，需要少量的DNA，需要大约4~5小时的动手工作。在所提出的技术中，DNA样本首先变性，然后杂交为以甲基化或参考位点的DNA为目标作为对照的探针。杂交DNA被分离成平行反应，一个只进行结扎，另一个进行结扎，随后在未甲基化GCGC序列下HhaI介导消化。由此产生的DNA片段被PCR扩增，并通过毛细血管电泳分离。HhaI 不消化甲基化 GCGC 位点并产生峰值信号，而未甲基化 GCGC 位点被消化，不生成峰值信号。比较每个样本的消化和未消化版本的控制-归一化峰可提供DNA样本的甲基化剂量比。在这里，MSPA用于检测骨肉瘤衍生的细胞外囊泡（EV）对间质干细胞中长穿插核元素1（LINE-1）甲基化状态的影响。LINE-1是重复性DNA元素，通常在癌症中进行低甲基化，以这种能力，可以作为生物标志物。超离心也被用作从生物液体中分离细胞外囊泡的具有成本效益的方法（即，在制备EV耗尽的胎儿牛血清[FBS]和从骨肉瘤调节介质中分离EV[差分离离]时）。对于甲基化分析，定制LINE-1探针设计用于针对LINE-1启动器序列中的三个甲基化位点和七个控制位点。该协议演示了使用MSPA进行LINE-1甲基化分析，并描述了通过超离心制备EV-耗尽FBS。

本研究的主要目的是评估OS-EV对MSCs的表观遗传效应。使用标准差分离离法从HOS-143B细胞中分离出OS-EV。典型的EV标记CD63，Hsp70和TSG101的西方印迹的表达证实了OS-EV的存在。（图2A）没有钙毒素信号表明OS-EV隔离物的纯度。使用 TEM 观察到了纯度的其他指示，存在各种尺寸的完整囊泡（图 2B）。OS-EV粒子平均浓度为7.63x1011/mL（...

Watch this Scientific Journal Video about LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles at JoVE.com

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

这里描述的是使用甲基化特异性探针扩增方法分析使用骨肉瘤衍生的细胞外囊泡治疗的中位体干细胞中LINE-1元素的甲基化水平。也演示了超中心化，一种从胎儿牛血清中分离细胞外囊泡的常用程序。

用骨肉瘤衍生细胞外囊泡处理的美相干细胞中的LINE-1甲基化分析

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

DNA甲基化是研究最多的表观遗传特征之一。许多癌症的特点是DNA甲基化水平的变化，这与异常基因表达和其他基因组异常有关。长穿插核元素是重复的可转置基因组序列，通常具有高水平的甲基化。然而，它们经常在癌症中变得高甲基化，导致它们的激活，并导致染色体不稳定性。在这项研究中，我们用骨肉瘤衍生的细胞外囊泡治疗间质干细胞，看看癌症 EV 是否会影响 MEC 中的 LINE-1 甲基化。胎儿牛血清是哺乳动物细胞培养基的一个组成部分，含有大量的牛源性电动汽车。这些 Evs 会干扰我们感兴趣的细胞对 Evs 的分析，因此，当生长用于 EV 隔离的细胞时，使用 EV 耗尽的 FBS 非常重要。将 FBS 放在超离心管中，并将它们放在超离心铲斗中。在将铲斗装入摆动转子之前，将铲斗平衡到 10 毫克内。将转子放在超中心，在10万G下运行19小时，温度为4摄氏度。运行后，小心地收集上清液的浅色上层，并转移到50毫升管。请勿打扰或移液深棕色颗粒，因为它含有来自 FBS 的 EV。通过 0.22 微米过滤器将上清液进入新的 50 毫升管中。这种过滤灭菌，EV耗尽的FBS现在可以添加到细胞培养时，生长细胞的EV隔离。从EV耗竭介质中生长的骨肉瘤细胞中收集条件介质。为了去除细胞和细胞碎片，在摄氏4度时将调节介质在2500 G下离心20分钟。将上流液转移到超离心管中，并像之前一样平衡它们。在摄氏4度下将管子在10万G下离心两小时。小心丢弃上清液，在底部留下约一毫升。将大约 20 毫升 PBS 加入管子中，轻轻移液，以便清洗和重新悬浮 EV 颗粒。平衡管子，并执行另一轮超离心与相同的设置。小心地去除上清液，通过温和移液将 EV 颗粒重新悬浮在 200 微升 PBS 中。将 EV 存放在低结合管中。纯化电动汽车的特点是西方印迹、纳米粒子跟踪分析和传输电子显微镜。24 井板中每井的板 15，000 MSC。24 小时后，取出旧介质，用 PBS 清洗细胞，然后更改为 EV 耗尽介质。在预定的时间点使用 OS-EV 处理细胞。停止EV治疗后，使用适当的方法从细胞中提取DNA。对于甲基化分析，我们使用了定制的甲基化特异性探针放大方法。设计定制的 LINE-1 探头，如前所述，由帕维契奇和其他人完成。对于甲基化探针，选择三个序列，其中包含启动子区域内的HhaI限制位点。对于控制探头，从 LINE-1 序列的其余部分中选择七个缺少 HhaI 限制站点的序列。将TE缓冲液中70纳米的DNA样本稀释至5微升。在 98 摄氏度下加热样品 10 分钟，然后冷却至 25 摄氏度。在每个样品中添加三个探针杂交混合微升，然后运行热循环器，使探头与DNA杂交。在室温下，在每个样品中加入30微升杂交后混合。将 10 微升转移到第二管。将两组管子放在热循环器中，并在 48 摄氏度下孵育至少一分钟。当样品在48摄氏度时，在第一组管子中加入10微升的结扎混合物，在第二组管子中加入10微升的结扎消化混合物。运行下一个热电塞程序。然后，向下旋转管，同时将热循环器设置为 72 摄氏度。在每个管中加入五升聚合酶混合物，并将管放在热循环器中。运行 PCR 程序。在 PCR 程序运行时，准备一毫升甲酰胺溶液，其中含有 2.5 微升尺寸标准。该解决方案的10微升管道，每排一排的光学96井板。PCR后，将未消化的样品和消化的样品稀释到超纯水中，分别稀释为一到一百和一至两百。在 96 井板中加入两个稀释 PCR 产品的微升。在 200 G 下将板离心 15 至 20 秒，以清除气泡。通过毛细管电泳对样品进行片段分析。打开毛细管电泳结果电图分析软件。选择一个示例，然后从菜单中将面板设置为 MLPA。单击面板并按控件 D 将此设置应用于所有样本。以类似的方式，将分析方法设置为所有样本的微卫星默认值。选择所有样本并单击绿色播放按钮以运行分析，然后选择所有样本并单击图形按钮以可视化探头峰值。放大峰值区域，获得单个峰值的更高分辨率。确保标记 LINE-1 探头组合中与探头对应的所有 10 个峰值。在基因型选项卡中，以 CSV 格式导出结果。在数据分析软件中打开 CSV 文件，将数据排序为列。根据三个甲基化站点的峰值大小对它们进行标记。其余七个峰值对应于控制探头。在这里，我们使用 LINE-1 2M 探针峰值，其大小约为 117 个基对。对于每个样本，计算所有七个控制峰值的总和峰值面积。将每个 LINE-1 探头的峰值区域除以此总和。对于每个样品，将消化样品的值除以未消化样品的值，以获得甲基化剂量比。西方印迹分析证实存在具有 TSG101、HSP70 和 CD63 信号的 OS-EV。缺乏卡内辛信号表明EV样品是纯的。使用 TEM 观察到额外的纯度指示，OS-EV 样品中存在各种尺寸的完整囊泡。通过NTA测量OS-EV颗粒浓度和大小分布。80% 的 EV 尺寸范围为 50 至 200 纳米。在这里，我们可以看到从MSC在EV治疗的不同时间点的S线-1的甲基化剂量比。灰线表示时间点零的基线甲基化水平。在时间点三，给药后平均甲基化剂量率下降，从而表示甲基化。EV 的这种超甲基化效应在时间点 7 时不太明显。在这里，我们展示了一种简单、高度敏感和稳健的甲基化分析技术。它需要少量的DNA，没有涉及双硫酸盐转化，它也适合从石蜡嵌入样品中分离的DNA。整个方法，与实验和分析部分，需要大约两天。由于基因组中 LINE-1 的拷贝数很高，因此 LINE-1 的基线甲基化在正常样本中很高。因此，与这种方法观察的样品甲基化水平的差异可能是微妙的。

Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

科研

遗传学

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...