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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| AGS cells | – | – | Gift from Christian Baron (Université de Montréal). |
| F12K Nut Mix 1X | GIBCO | 21127022 | Culturing medium for AGS cells |
| L-Glutamine | CORNING | MT25005CI | Component of cell culturing medium |
| Penicillin Streptomycin Solution | CORNING | 30-002-CI | Component of cell culturing medium |
| Fetal Bovine Serum | Sigma Aldrich | F2442 | Component of cell culturing medium |
| DMEM 1X | GIBCO | 21068028 | culturing medium for phoenix cell |
| CaCl2 | Sigma Aldrich | C1016 | used in phoenix cell transfection |
| HEPES | Sigma Aldrich | H3375 | used in phoenix cell transfection (HBS solution) |
| KCl | Sigma Aldrich | P9333 | used in phoenix cell transfection (HBS solution) |
| Dextrose | Sigma Aldrich | D9434 | used in phoenix cell transfection (HBS solution) |
| NaCl | Sigma Aldrich | S7653 | used in phoenix cell transfection (HBS solution) and retrovirus precipitation |
| Na2HPO4 | Sigma Aldrich | S3264 | used in phoenix cell transfection (HBS solution) |
| TRYPSIN EDTA SOLUTION 1X | CORNING | 59430C | used in cell split |
| DPBS 1X | GIBCO | 14190250 | Dulbecco's Phosphate Buffered Saline |
| DMSO | Sigma Aldrich | D8418 | Component of cell freezing medium (80% FBB and 20% DMSO) |
| G-418 Disulphate | Formedium | G4185 | selection drug for |
| Gelatin solution Bioreagent | Sigma Aldrich | G1393 | cotaing of 96 well DNA plate and freezing plate |
| Tris-base | Fisher BioReagents | 10376743 | Component of Cell lysis buffer for genomic DNA extraction |
| EDTA | Sigma Aldrich | E6758 | Component of Cell lysis buffer for genomic DNA extraction |
| SDS | Sigma Aldrich | 71729 | Component of Cell lysis buffer for genomic DNA extraction |
| Proteinase K | Thermo Fisher | AM2546 | Component of Cell lysis buffer for genomic DNA extraction |
| RNAse A | Thermo Fisher | 12091021 | RNA degradation during DNA extraction |
| Agarose | Sigma Aldrich | A5304 | DNA gel preparation |
| Atlas ClearSight | Bioatlas | BH40501 | Stain reagent used for detecting DNA and RNA samples in agarose gel |
| ethanol | Fisher BioReagents | BP28184 | DNA precipitation |
| Sodium Acetate | Sigma Aldrich | 71196 | Used for DNA precipitation at a 3M concentration pH5.2 |
| Wizard SV Gel and PCR clean-Up system | Promega | A9282 | Extraction of PCR fragments from agarose gel during PCR screening of neomycin positive clones |
| Trizol | AMBION | 15596018 | Organic solvent used for RNA extraction |
| Dnase I | THERMO SCIENTIFIC | 89836 | degradation of genomic DNA from RNA |
| dNTPs mix | Invitrogen | 10297018 | used in RT and PCR reactions |
| DTT | Invitrogen | 707265ML | used in RT reactions |
| diethyl pyrocarbonate | Sigma Aldrich | D5758 | used to inactivate RNAses in water (1:1000 dilution) |
| Ribolock | Thermo Fisher | EO0381 | RNase inhibitor |
| MOPS | Sigma Aldrich | M9381 | preparation of RNA gel |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | preparation of denaturating RNA gel (1% PFA in 1x MOPS) |
| Superscript III Reverse transcriptase | Invitrogen | 18080-093 | Retrotranscription reaction |
| Pfu DNA polymerase (recombinant) | Thermo Scientific | EP0501 | PCR reaction |
| 2X qPCRBIO SyGreen Mix Separate-ROX | PCR BIOSYSTEMS | PB 20.14 | qPCR reaction |
| Cre-GFP adenovirus | https://medicine.uiowa.edu/vectorcore | 1174-HT | used to infect TERRA-MS2 clones in order to remove the neomycn gene |
| Sodium Butyrate | Sigma Aldrich | B5887 | used to promote retrovirus particles production in phoenix cells |
| PEG8000 | Sigma Aldrich | 89510 | Precipitation of retrovirus partcles |
| 35µ-Dish Glass Bottom | Ibidi | 81158 | used in live cell imaging analyses of TERRA-MS2 clones |
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