Name: generation of cancer cell clones to visualize telomeric repeat-containing rna terra expressed from a single telomere in living cells
Uploaded: 2019-01-17T14:30:00
Description: Watch this Scientific Journal Video about Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells at JoVE.com

我们感谢特伦托大学 ciboi 先进成像设施的工作人员和维也纳 max f. perutz 实验室 (mfpl) 的生物光学光学显微镜设施的工作人员。导致这些成果的研究得到了 Mahlke-Obermann 基金会和欧洲联盟根据609431欧盟赠款协议进行的第七个研究、技术开发和示范框架方案的资助。欧共体得到意大利教育大学和研究部 (miur) 的 rita levi montalcini 研究金的支持。

(一)新霉素耐药克隆的选择
<ol><li>在哈姆赫-12k (kaighn) 培养基中生长 ags 细胞, 辅以10% 的胎儿牛血清 (fbs)、2 mm l-谷氨酰胺、青霉素 (培养基每毫升0.5 单位) 和链霉素 (每毫升介质 0.2μg) 和 5% co2.将细胞与 sgrnams9 表达载体和 ms2 盒式磁带以50-60 的融合方式转染, 以1:10 摩尔比21。 注: 在并行实验中, 通过转染 transfection 表达向量 (即cas9-gfp 矢量) 来验证转染效率。应达到...

<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=Developmentally+regulated+transcription+of+mammalian+telomeres+by+DNA-dependent+RNA+polymerase+II.">Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II.</a> Nature Cell Biology. 10 (2), 228-236 (2008).</li><li>Diman, A., Decottignies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+origin+and+nuclear+localization+of+TERRA+telomeric+repeat-containing+RNA:+from+Darkness+to+Dawn.">Genomic origin and nuclear localization of TERRA telomeric repeat-containing RNA: from Darkness to Dawn.</a> FEBS Journal. , (2017).</li><li>Arnoult, N., Van Beneden, A., Decottignies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+regulates+TERRA+levels+through+increased+trimethylation+of+telomeric+H3K9+and+HP1alpha.">Telomere length regulates TERRA levels through increased trimethylation of telomeric H3K9 and HP1alpha.</a> Nature Structural &amp; Molecular Biology. 19 (9), 948-956 (2012).</li><li>Montero, J. J., 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=TERRA+recruitment+of+polycomb+to+telomeres+is+essential+for+histone+trymethylation+marks+at+telomeric+heterochromatin.">TERRA recruitment of polycomb to telomeres is essential for histone trymethylation marks at telomeric heterochromatin.</a> Nature Communications. 9 (1), 1548 (2018).</li><li>Beishline, K., 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=CTCF+driven+TERRA+transcription+facilitates+completion+of+telomere+DNA+replication.">CTCF driven TERRA transcription facilitates completion of telomere DNA replication.</a> Nature Communications. 8 (1), 2114 (2017).</li><li>Arora, 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=RNaseH1+regulates+TERRA-telomeric+DNA+hybrids+and+telomere+maintenance+in+ALT+tumour+cells.">RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells.</a> Nature Communications. 5, 5220 (2014).</li><li>Balk, 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=Telomeric+RNA-DNA+hybrids+affect+telomere-length+dynamics+and+senescence.">Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence.</a> Nature Structural &amp; Molecular Biology. 20 (10), 1199-1205 (2013).</li><li>Graf, 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=Telomere+Length+Determines+TERRA+and+R-Loop+Regulation+through+the+Cell+Cycle.">Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle.</a> Cell. 170 (1), 72-85 (2017).</li><li>Lee, Y. W., Arora, R., Wischnewski, H., Azzalin, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF1+participates+in+chromosome+end+protection+by+averting+TRF2-dependent+telomeric+R+loops.">TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.</a> Nature Structural &amp; Molecular Biology. , (2018).</li><li>Moravec, 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=TERRA+promotes+telomerase-mediated+telomere+elongation+in+Schizosaccharomyces+pombe.">TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.</a> EMBO Reports. 17 (7), 999-1012 (2016).</li><li>Cusanelli, E., Romero, C. A., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+noncoding+RNA+TERRA+is+induced+by+telomere+shortening+to+nucleate+telomerase+molecules+at+short+telomeres.">Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.</a> Molecular Cell. 51 (6), 780-791 (2013).</li><li>Moradi-Fard, S., 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=Smc5/6+Is+a+Telomere-Associated+Complex+that+Regulates+Sir4+Binding+and+TPE.">Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE.</a> PLoS Genetics. 12 (8), e1006268 (2016).</li><li>Chu, H. 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=TERRA+RNA+Antagonizes+ATRX+and+Protects+Telomeres.">TERRA RNA Antagonizes ATRX and Protects Telomeres.</a> Cell. 170 (1), 86-101 (2017).</li><li>Lai, L. T., Lee, P. J., Zhang, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+protects+RNA+signals+in+simultaneous+RNA-DNA+FISH.">Immunofluorescence protects RNA signals in simultaneous RNA-DNA FISH.</a> Experimental Cell Research. 319 (3), 46-55 (2013).</li><li>Zhang, L. F., 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=Telomeric+RNAs+mark+sex+chromosomes+in+stem+cells.">Telomeric RNAs mark sex chromosomes in stem cells.</a> Genetics. 182 (3), 685-698 (2009).</li><li>Gallardo, F., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+mRNAs+in+fixed+and+living+yeast+cells.">Visualizing mRNAs in fixed and living yeast cells.</a> Methods in Molecular Biology. 714, 203-219 (2011).</li><li>Querido, E., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescent+proteins+to+study+mRNA+trafficking+in+living+cells.">Using fluorescent proteins to study mRNA trafficking in living cells.</a> Methods in Cell Biology. 85, 273-292 (2008).</li><li>Cusanelli, E., Chartrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+noncoding+RNA:+telomeric+repeat-containing+RNA+in+telomere+biology.">Telomeric noncoding RNA: telomeric repeat-containing RNA in telomere biology.</a> Wiley Interdisciplinary Reviews: RNA. 5 (3), 407-419 (2014).</li><li>Perez-Romero, C. A., Lalonde, M., Chartrand, P., Cusanelli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+relocalization+of+telomeric+repeat-containing+RNAs+during+diauxic+shift+in+budding+yeast.">Induction and relocalization of telomeric repeat-containing RNAs during diauxic shift in budding yeast.</a> Current Genetics. , (2018).</li><li>Avogaro, L., 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=Live-cell+imaging+reveals+the+dynamics+and+function+of+single-telomere+TERRA+molecules+in+cancer+cells.">Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells.</a> RNA Biology. , 1-10 (2018).</li><li>Ran, F. 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=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154 (6), 1380-1389 (2013).</li><li>Yamada, T., 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=Spatiotemporal+analysis+with+a+genetically+encoded+fluorescent+RNA+probe+reveals+TERRA+function+around+telomeres.">Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.</a> Scientific Reports. 6, 38910 (2016).</li><li>Deng, Z., 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=Formation+of+telomeric+repeat-containing+RNA+(TERRA)+foci+in+highly+proliferating+mouse+cerebellar+neuronal+progenitors+and+medulloblastoma.">Formation of telomeric repeat-containing RNA (TERRA) foci in highly proliferating mouse cerebellar neuronal progenitors and medulloblastoma.</a> Journal of Cell Science. 125 (Pt 18), 4383-4394 (2012).</li><li>Casini, 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=A+highly+specific+SpCas9+variant+is+identified+by+in+vivo+screening+in+yeast.">A highly specific SpCas9 variant is identified by in vivo screening in yeast.</a> Nature Biotechnology. 36 (3), 265-271 (2018).</li><li>Nergadze, S. 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在本文中, 我们提出了一种方法来生成人类癌症细胞克隆包含 ms2 序列集成在亚端粒15q。利用这些克隆体, 用荧光显微镜通过 ms2-gfp 融合蛋白的共表达检测从近端粒15q 转录的 ms2 标记的 terra 分子。这种方法使研究人员能够研究在活细胞21中的单个端粒表达的 terra 的动力学。在该协议中, 在 ags 细胞系中选择 terra-ms2 克隆, 这代表了一个有趣的模型系统来研究 terra, 因为 terra 表达在人?...

长非编码 rna terra 从染色体的亚端粒区域转录, 其转录向染色体末端进行, 在端粒重复道1,2内终止。因此, terra 记录由 5 ' 端的亚端粒衍生序列组成, 并以端粒重复终止 (脊椎动物中的 uuaggg)3。提出了 terra 的重要作用, 包括端粒4、5、dna 复制 6的异染色质形成, 促进染色体末端 7,8 的同源重组,9、调节端粒结构10和端粒长度稳态2,11,12,13。此外, terra 转录与许多体外位点相互作用, 以调节小鼠胚胎干细胞 (es) 中广泛的基因表达。根据这些证据, rna荧光原位杂交 (rna-fish) 分析表明, 只有一部分 terra 转录记录定位端粒1,2,15。此外, 据报道, terra 在小鼠细胞2,16的 x 和 y 染色体处形成核聚集物。这些发现表明, terra 记录在细胞核内经历了复杂的动态。了解 terra 分子的动力学将有助于确定其功能和作用机制。
ms2-gfp 系统已被广泛用于可视化来自不同生物体的活细胞中的 rna 分子 17,18.该系统以前曾被用来标记和可视化单端粒 terra 分子在酿酒师12,19。利用该系统, 最近显示酵母 terra 转录转录位在细胞质内的后二氧化转变阶段, 这表明 terra 可能会发挥核外功能20。我们最近使用 ms2-gfp 系统研究癌细胞21中的单端粒 terra 转录.为此, 我们使用 crispr/cas9 基因组编辑工具将 ms2 序列集成在单个端粒 (端粒 15q, 以下为 telq 15q) 上, 并获得了表示 ms2 标记内生 t15q terra (terra-ms2 克隆) 的克隆。gfp 熔融 ms2 rna 结合蛋白 (ms2-gfp) 的共表达, 可识别并结合 ms2 rna 序列, 从而实现活细胞21中单端粒 terra 转录的可视化.本文所述协议的目的是详细描述生成 terra-ms2 克隆所需的步骤。
为了生成 terra-ms2 克隆, ms2 盒被集成在端粒15q 的亚端粒区域内, 位于 terra 启动子区域和转录起始位点的下游。ms2 盒式磁带含有由 lox-p 位点两侧的新霉素耐药基因, 其在近端粒15q 上的整合是使用 crispr/cas9 系统22进行的。经转染 ms2 盒后, 选择单克隆, 用 pcr、dna 测序和南方印迹验证盒的亚端粒积分。阳性克隆感染了表达 crec 的腺病毒, 以便删除盒式磁带中的选择标记, 只在近端粒15q 留下一个 ms2 序列和一个 lox-p 位点。通过 rt-qpcr 验证了 tel15q 中 ms2 标记的 terra 转录的表达。最后, ms2-gfp 融合蛋白通过逆转录病毒感染在 terra-ms2 克隆中表达, 以便用荧光显微镜显示 ms2-terra 转录。使用端粒重复特异性探针 1、2、15、23可通过 rna-fish 和活细胞成像快速检测 terra 记录。这些方法提供了关于 terra 分子在单个细胞分辨率下的总种群定位的重要信息。在单个亚端粒生成含有 ms2 序列的克隆, 将使研究人员能够研究活细胞中单端粒 terra 转录的动力学, 这将有助于确定 terra 的功能和作用机制。

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	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:245px;">AGS cells</td>
			<td style="width:246px;">-</td>
			<td style="width:105px;">-</td>
			<td style="width:583px;">Gift from Christian Baron (Universit&#233; de Montr&#233;al).</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">F12K Nut Mix 1X</td>
			<td>GIBCO</td>
			<td>21127022</td>
			<td>Culturing medium for AGS cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-Glutamine&#160;</td>
			<td>CORNING</td>
			<td>MT25005CI</td>
			<td>Component of cell culturing medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Penicillin Streptomycin Solution</td>
			<td>CORNING</td>
			<td>30-002-CI</td>
			<td>Component of cell culturing medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>Component of cell culturing medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM 1X</td>
			<td>GIBCO</td>
			<td>21068028</td>
			<td>culturing medium for phoenix cell</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C1016</td>
			<td>used in phoenix cell transfection</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>D9434</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>used in phoenix cell transfection (HBS solution) and retrovirus precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Na2HPO4</td>
			<td>Sigma Aldrich</td>
			<td>S3264</td>
			<td>used in phoenix cell transfection (HBS solution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">TRYPSIN EDTA SOLUTION 1X</td>
			<td>CORNING</td>
			<td>59430C</td>
			<td>used in cell split</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DPBS 1X</td>
			<td>GIBCO</td>
			<td>14190250</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D8418</td>
			<td>Component of cell freezing medium (80% FBB and 20% DMSO)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:245px;">G-418 Disulphate</td>
			<td>Formedium</td>
			<td>G4185</td>
			<td>selection drug for&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gelatin solution Bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>cotaing of 96 well DNA plate and freezing plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tris-base</td>
			<td>Fisher BioReagents</td>
			<td>10376743</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SDS</td>
			<td>Sigma Aldrich</td>
			<td>71729</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Proteinase K</td>
			<td>Thermo Fisher</td>
			<td>AM2546</td>
			<td>Component of Cell lysis buffer for genomic DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">RNAse A</td>
			<td>Thermo Fisher</td>
			<td>12091021</td>
			<td>RNA degradation during DNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A5304</td>
			<td>DNA gel preparation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Atlas ClearSight</td>
			<td>Bioatlas</td>
			<td>BH40501</td>
			<td>Stain reagent used for detecting DNA and RNA samples in agarose gel</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ethanol</td>
			<td>Fisher BioReagents</td>
			<td>BP28184</td>
			<td>DNA precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium Acetate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>71196</td>
			<td>Used for DNA precipitation at a 3M concentration pH5.2</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wizard SV Gel and PCR clean-Up system</td>
			<td>Promega</td>
			<td>A9282</td>
			<td>Extraction of PCR fragments from agarose gel during PCR screening of neomycin positive clones</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trizol</td>
			<td>AMBION</td>
			<td>15596018</td>
			<td>Organic solvent used for RNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dnase I</td>
			<td>THERMO SCIENTIFIC</td>
			<td>89836</td>
			<td>degradation of genomic DNA from RNA&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">dNTPs mix</td>
			<td>Invitrogen</td>
			<td>10297018</td>
			<td>used in RT and PCR reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DTT</td>
			<td>Invitrogen</td>
			<td>707265ML</td>
			<td>used in RT reactions</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">diethyl pyrocarbonate</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>used to inactivate RNAses in water (1:1000 dilution)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ribolock</td>
			<td>Thermo Fisher</td>
			<td>EO0381</td>
			<td>RNase inhibitor</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M9381</td>
			<td>preparation of RNA gel</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>preparation of denaturating RNA gel (1% PFA in 1x MOPS)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Superscript III Reverse transcriptase</td>
			<td>Invitrogen</td>
			<td>18080-093</td>
			<td>Retrotranscription reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pfu DNA polymerase (recombinant)</td>
			<td>Thermo Scientific</td>
			<td>EP0501</td>
			<td>PCR reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2X qPCRBIO SyGreen Mix Separate-ROX</td>
			<td>PCR BIOSYSTEMS</td>
			<td>PB 20.14</td>
			<td>qPCR reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cre-GFP adenovirus</td>
			<td>https://medicine.uiowa.edu/vectorcore</td>
			<td>1174-HT</td>
			<td>used to infect TERRA-MS2 clones in order to remove the neomycn gene</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium Butyrate</td>
			<td>Sigma Aldrich</td>
			<td>B5887</td>
			<td>used to promote retrovirus particles production in phoenix cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG8000</td>
			<td>Sigma Aldrich</td>
			<td>89510</td>
			<td>Precipitation of retrovirus partcles</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">35&#181;-Dish Glass Bottom</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td>used in live cell imaging analyses of TERRA-MS2 clones</td>
		</tr>
	</tbody>
</table>

generation of cancer cell clones to visualize telomeric repeat-containing rna terra expressed from a single telomere in living cells

端粒被转录, 导致端粒重复编码 rna (terra), 这已被建议在端粒生物学中发挥重要作用, 包括异染色质的形成和端粒长度的稳态。最近的发现表明, terra 分子也与内部染色体区域相互作用, 以调节小鼠胚胎干细胞 (es) 细胞的基因表达。根据这一证据, rna 荧光原位杂交 (rnaa-fish) 分析表明, 只有一部分 terra 转录记录定位在染色体末端。更好地了解 terra 分子的动力学将有助于确定其功能和作用机制。在这里, 我们描述了一种方法, 标签和可视化单端粒 terra 转录在癌细胞使用 ms2-gfp 系统。为此, 我们提出了一个协议, 以产生稳定的克隆, 使用 ags 人类胃癌细胞系, 包含 ms2 序列集成在一个单一的亚端粒。从 ms2 标记的端粒中转录 terra 的结果是 ms2 标记的 terra 分子的表达, 这些分子在 ms2 rna 结合蛋白融合蛋白与 gfp (ms2-gfp) 共表达后, 通过活细胞荧光显微镜进行了可视化。这种方法使研究人员能够研究癌细胞中单端粒 terra 分子的动力学, 并可应用于其他细胞系。

图 11表示实验策略的概述。该协议的主要步骤和在 ags 单元中生成 terra-ms2 克隆的指示性时间表如下图所示 (图 1a)。在第1天, 用 ms2 盒式磁带和 sgrna/cas9 表示向量转染6孔的多个井 (如图 1b 所示)。在 cas9 nickase 表示 px335 载体中克隆了两个不同的近端粒15q 特异性引导 rna 序列,...

Watch this Scientific Journal Video about Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells at JoVE.com

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

在这里, 我们提出了一个协议, 以生成包含 ms2 序列标记在单个亚端粒的癌细胞克隆。这种方法, 依靠 ms2-gfp 系统, 实现了端粒重复 rna (terra) 的内源性转录记录的可视化表达从一个端粒表达在活细胞中。

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

这种方法可以帮助回答有关端粒的关键问题，如酵母端粒非编码RNATERRA本地化，我可以坚持它是端粒的起源或跨，这种技术的主要优点是，它允许研究人员可视化TERRA分子从活细胞中的单个端粒表达。演示这个程序的将是克劳迪奥·奥斯·佩戈拉尔和妮可·贝廷，两个研究生从我的实验室。根据文本协议开始生长AGS细胞。然后，当细胞达到50-60%的汇合时，用sgRNA Cas9表达向量和MS2盒。第二天，用含有Neomycin的培养介质取代栽培介质。在此之后，在 96 井板的每个井中加入 10 微升 0.25% 的 Tripsyn。使用显微镜和标记标记盘中可见的每个克隆的位置。用足够的PBS取代Neomycin培养介质，在克隆上形成一层液体薄膜。使用含有5微升的尝试辛的10微升移液器，慢慢释放尝试素到殖民地。允许 Trypsin 分离单元格一分钟。用尖端刮掉殖民地，然后吸进尖端。在此之后，将细胞从菌落转移到96井板的井中。在室温下孵育板五分钟。然后用150微升的选择性介质填充井。接下来，在三个96孔板的每个井中加入100微升明胶。在室温下孵育板30分钟，用PBS清洗板两次。克隆达到90%汇合后，从板的每个井中吸出介质，用PBS清洗板材。然后在每个井中加入30微升的 Trypsin，并在37摄氏度下孵育板5分钟。接下来，在每个油井中加入70微升选择性介质。通过上下移液来破坏细胞。将混合物的30微升转移到含有120微升选择性介质的明胶化DNA板的井中。然后将混合物的30微升转移到含有50微升介质的明化冷冻板。将DNA放在培养箱中，让细胞在37摄氏度下生长，直到达到90%的汇合。在此之后，在冷冻板的每个井中加入80微升的冰冷介质。用副膜密封板，更换盖子，用铝箔包裹板。然后将冻结板保持在 80 摄氏度。DNA板中的克隆达到90%汇合后，用PBS清洗两次。然后用50微升的Lysis缓冲液来解化细胞。用副膜盖住盘子，更换盖子，然后用萨兰包装。在此之后，在37摄氏度的温度下孵育板。第二天，在每一个井中加入100微升的乙醇氯化钠溶液，并允许DNA沉淀6小时或在室温下过夜。然后倒置盘子，用200微升乙醇洗三次，每井70%。在板的每个井中加入20微升的RNase A溶液，并在37摄氏度下孵育板一小时。之后，使用三个微升的基因组DNA进行PCR扩增，并设置PCR机。根据文本协议，在六个孔板中生长 PCR 正克隆。一旦克隆达到90%的汇合，用PBS清洗细胞，并添加250微升的Lysis缓冲液与蛋白酶K到每个井。使用细胞刮刀刮取细胞，将 Lysate 转移到 1.5 毫升管中。在37摄氏度下孵育Lysate16小时。在 Lysate 中加入一毫升乙醇，然后剧烈摇晃。允许克隆在 F-12K 介质中根据文本协议增长。然后，一旦细胞达到70%的汇合，再将克里表达腺病毒加入细胞。感染48小时后，将细胞分成三个10厘米的盘子。然后培养第三道菜细胞冷冻，和RNA提取。首先，在玻璃底盘中盘制TERRA-MS2克隆和野生型AGS细胞。然后添加多聚氨酯和 MS2-GFP 表达逆转录病毒到介质。24小时后丢弃含有逆转录病毒的介质，并在细胞中加入不含酚红的新鲜介质。最后，使用倒置显微镜和灵敏的相机图像对具有适当目标和光圈的细胞进行图像。在此协议中，在单个子电信体上创建并成像包含 MS2 序列标记的癌细胞克隆。使用PCR对抗新霉素克隆进行筛选。阳性克隆在细胞培养中培养，用于基因组DNA提取和南方印迹分析。对PCR和南方印迹分析呈阳性的克隆人感染了CRE-GFP表达腺病毒，以去除MS2盒中存在的Neomycin基因。一旦新霉素抗药基因的消除得到确认，从克隆中提取的RNA被测试为TERRA-MS2转录本的表达。为了通过共和显微镜在活细胞中可视化TERRA-MS2转录，选定的克隆人感染了表达MS2-GFP融合蛋白的逆转录病毒。TERRA-MS2转录本和端粒通过共生显微镜同时在活细胞中可视化，通过共同表达MS2-GFP融合蛋白和在选定克隆中融合端粒结合蛋白TRF2。尝试此过程时，必须尽一切努力选择单个克隆，而不是混合克隆。按照这个程序，其他方法，如DNA FISH在染色体传播，可用于可视化的MS2盒在固定细胞的集成。该技术开发后，可为端粒领域的研究人员探索活体细胞中单端粒TERRA分子的细胞定位铺平道路。不要忘记，使用病毒载体和放射性物质可能是极其不确定的。执行此过程时，应始终采取预防措施，例如穿着实验室外套、手套和使用有机玻璃护罩以尽量减少辐射。

Research

JoVE Journal

Genetics

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 ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...