方法文章

通过单个端粒可视化活细胞中端粒重复序列包含RNA（TERRA）的癌细胞克隆构建

DOI：

10.3791/58790

⸱

2019年1月17日

 ,  ,  , 
1Laboratory of Cell Biology and Molecular Genetics, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, 2Department of Life Sciences, University of Trieste

本文内容

摘要

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本文介绍一种在单个亚端粒区域插入MS2序列标签以生成癌细胞克隆的实验方案。该方法基于MS2-GFP系统，可在活细胞中实现对单个端粒来源的端粒重复序列包含RNA（TERRA）内源性转录本的可视化观察。

摘要

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端粒被转录产生含有端粒重复序列的长链非编码RNA（TERRA），据认为其在端粒生物学中发挥重要作用，包括异染色质形成和端粒长度稳态的调控。最新研究发现，TERRA分子还可与染色体内部区域相互作用，从而调控小鼠胚胎干细胞（ES细胞）中的基因表达。与此证据一致，RNA荧光 原位 杂交（RNA-FISH）分析表明，仅有一部分TERRA转录本定位于染色体末端。深入理解TERRA分子的动态行为将有助于阐明其功能及作用机制。本文介绍了一种利用MS2-GFP系统在癌细胞中标记并可视化单个端粒TERRA转录本的方法。为此，我们提供了一项实验方案，以AGS人胃癌细胞系为例，构建稳定克隆，使其在单一亚端粒区整合MS2序列。当带有MS2标签的端粒转录产生TERRA时，会表达出带有MS2标签的TERRA分子；通过共表达与GFP融合的MS2 RNA结合蛋白（MS2-GFP），可在活细胞荧光显微镜下观察到这些分子。该方法使研究人员能够研究癌细胞中单个端粒来源TERRA分子的动态变化，并可推广应用于其他细胞系。 关键词：TERRA转录本，端粒，MS2-GFP系统，活细胞成像，RNA-FISH，AGS细胞系，染色体末端，单分子标记

引言

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长链非编码RNA TERRA从染色体的端粒近端区域转录，其转录方向朝向染色体末端，并在端粒重复序列区域内终止1,2因此，TERRA 转录本的5'端包含由亚端粒区衍生而来的序列，并以端粒重复序列（在脊椎动物中为UUAGGG）结尾3TERRA 被认为在端粒处的异染色质形成中发挥重要作用4,5DNA复制6促进染色体末端之间的同源重组7,8,9，调控端粒结构10端粒长度稳态2,11,12,13此外，TERRA 转录本与小鼠胚胎干细胞（ES 细胞）中大量端粒外位点相互作用，从而调控广泛的基因表达14与这些证据一致，RNA 荧光 原位 杂交（RNA-FISH）分析表明，仅有一部分TERRA转录本定位于端粒1,2,15此外，有报道称TERRA可在小鼠细胞中形成定位于X和Y染色体的核内聚集体2,16这些发现表明，TERRA 转录本在细胞核内经历复杂的动态变化。解析 TERRA 分子的动态特性将有助于阐明其功能及作用机制。

MS2-GFP 系统已被广泛用于在多种生物的活细胞中可视化 RNA 分子17,18。该系统先前已被用于标记并可视化酿酒酵母（S. cerevisiae）中的单个端粒 TERRA 分子12,19。利用该系统，最近的研究发现酵母 TERRA 转录本在双相生长转换后期定位于细胞质中，提示 TERRA 可能具有细胞核外功能20。我们近期已使用 MS2-GFP 系统在癌细胞中研究单个端粒的 TERRA 转录本21。为此，我们采用 CRISPR/Cas9 基因组编辑技术将 MS2 序列整合至单个端粒（15号染色体长臂端粒，以下简称 Tel15q），并获得了表达 MS2 标记的内源性 Tel15q TERRA 的克隆（TERRA-MS2 克隆）。通过共表达一种与 GFP 融合的 MS2 RNA 结合蛋白（MS2-GFP），该蛋白可识别并结合 MS2 RNA 序列，从而实现在活细胞中对单个端粒 TERRA 转录本的可视化21。本方案的目的是详细描述生成 TERRA-MS2 克隆所需的各个步骤。

为构建TERRA-MS2克隆，需将MS2表达盒整合至15q端粒的亚端粒区，位于TERRA启动子区域及其转录起始位点的下游。该MS2表达盒包含一个两侧带有lox-p位点的新霉素抗性基因，其在15q亚端粒区的整合通过CRISPR/Cas9系统完成22。转染MS2表达盒后，筛选单克隆，并通过PCR、DNA测序和Southern印迹法验证表达盒在亚端粒区的正确整合。对阳性克隆感染表达Cre重组酶的腺病毒，以去除表达盒中的筛选标记，仅保留MS2序列和一个lox-p位点于15q亚端粒区。通过RT-qPCR验证来自Tel15q的MS2标记TERRA转录本的表达。最后，通过逆转录病毒感染在TERRA-MS2克隆中表达MS2-GFP融合蛋白，从而利用荧光显微镜观察MS2标记的TERRA转录本。使用端粒重复序列特异性探针，可通过RNA-FISH和活细胞成像清晰检测到TERRA转录本1,2,15,23。这些方法可在单细胞分辨率下提供TERRA分子总体定位的重要信息。在单一亚端粒区域含有MS2序列的克隆构建成功后，研究人员将能够研究活细胞中单个端粒来源的TERRA转录本的动态行为，从而有助于阐明TERRA的功能及其作用机制。

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方案

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1 . Selection of Neomycin Resistant Clones

  1. Grow AGS cells in Ham's F-12K (Kaighn's) medium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, penicillin (0.5 units per mL of medium), and streptomycin (0.2 µg per mL of medium) at 37 °C and 5% CO2. Transfect the cells at a 50-60% confluence with the sgRNA/Cas9 expressing vector and the MS2 cassette at a 1:10 molar ratio21.
    NOTE: In a parallel experiment, verify transfection efficiency by transfecting a GFP-expressing vector (i.e., Cas9-GFP vector). At least 60-70% transfection efficiency should be achieved.
  2. The following day, replace the culturing medium with medium containing neomycin at 0.7 µg/mL final concentration (selective medium).
    NOTE: Splitting the cells and seeding them in selective medium the day after transfection will speed up the selection process. All non-transfected cells will immediately die. If transfection is performed in 6 well plates, in which case each well at a 60% confluence would contain approximately 0.7 x 106 cells, on the following day the cells can be split from a single well to a 10 cm dish containing selective medium.
  3. Keep the cells in selective medium for 7-10 days, changing medium every one or two days, until single clones are visible.
  4. Picking of cell clones
    1. Prepare a 96 well plate containing 10 µL of 0.25% trypsin in each well.
      NOTE: Prepare two 96 well plates in case more than 96 clones are expected to be picked.
    2. With the use of a microscope, mark the position of each clone visible in the 10 cm dish by making a dot at the bottom of the dish using a marker. Each dot will correspond to a colony to be picked.
    3. Replace the culturing medium with just enough Phosphate Buffered Saline (PBS) to form a thin film of liquid on the clones and not let the cells dry during the clone picking.
    4. Pick single colonies using a 10 µL pipette. Attach the tip containing 5 µL of trypsin to the colony and slowly release the trypsin which will remain localized on the colony. Allow trypsin to detach the cells for 1 min, then scrape the colony with the tip and suck it up into the tip.
      NOTE: During this procedure, flipping the dish a bit on one side so to decrease the volume of PBS around the colony being picked will help the picking process by avoiding diluting the trypsin around the colony. Using a clone ring may also help picking up single clones.
    5. Place the cells from the colony into a well of the 96 well plate containing 10 µL of 0.25% trypsin.
    6. Incubate 5 min at room temperature, then fill the well with 150 µL of selective medium (Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 µg per mL of medium) and containing neomycin at a concentration of 0.7 µg/mL.
      NOTE: During the incubation time other clones can be picked. It is recommended to pick as many clones as possible. The more clones are picked the higher the chances will be of identifying positive ones.
    7. Once all the clones are picked and transferred in the 96 well plate, allow the cells to grow for a few days in selective medium Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 µg per mL of medium) and containing neomycin at a concentration of 0.7 µg/mL at 37 °C and 5% CO2, until reaching 90% confluence.
  5. Splitting of clones
    1. Prepare three 96 well plates coated with gelatin by adding 100 µL of gelatin per well, incubate for 30 min at room temperature, then wash two times with PBS. These plates will be used for DNA extraction (DNA plate) and for clone freezing (freezing plates).
      NOTE: Gelatin will promote the attachment of the cells and of the DNA to the wells. In particular, the gelatin coating will allow the DNA to stick at the bottom of the wells during the DNA extraction and wash procedures (discussed below). During the clone-splitting procedure, it is advisable to use a multichannel pipette.
    2. Once clones reach 90% confluence, aspirate medium from each well of the 96 well plate, wash with PBS, add 30 µL of 0.25% trypsin per well, and incubate for 5 min at 37 °C.
      NOTE: Clones will grow at different rates, which will also depend on the number of cells picked per clone. Thus, this step will be performed during the several days when the different clones reach 90% confluence.
    3. Add 70 µL of selective medium per well, disrupt cell clumps by pipetting up and down inside the wells, then transfer 30 µL of the 100 µL to the gelatinized DNA plate prefilled with 120 µL of selective medium per well and 30 µL to the gelatinized freezing plates prefilled with 50 µL medium (without selection).
    4. Place the DNA plate in the incubator and allow the cells to grow at 37 °C and 5% CO2 until 90% confluence.
    5. Add 80 µL of ice-cold freshly made 2x freezing medium (80% FBS and 20% dimethyl sulfoxide (DMSO)) to each well of the freezing plate, add parafilm (sprayed with 70% ethanol) on top of the plate so to seal each well, place the lid on top and wrap the plate with aluminum foil. Place the freezing plates at -80 °C.
    6. Add 90 µL of selective medium per well to the original 96 well plate containing the clones that have been split and keep it in culture until PCR screening results. This plate will be used as backup plate.

2. Screening of Neomycin Resistant Clones

  1. DNA extraction from the 96 well DNA plate.
    1. Once the clones cultured in the DNA plate reach 90% confluence, wash 2 times with PBS, then lyse with 50 µL lysis buffer (10 mM Tris pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, and 1 mg/mL proteinase K). Cover the plate with parafilm, sealing each well, put the lid on, cover with saran wrap, and place at 37 °C overnight.
    2. Add 100 µL of cold ethanol (Et-OH)/NaCl solution (0.75 M NaCl in 100% ethanol) to each well and precipitate for 6 h or overnight at room temperature.
      NOTE: The protocol can be paused here.
    3. Remove the Et-OH/NaCl solution by inverting the plate and wash 3 times with 200 µL of 70% ethanol per well.
    4. Add 25 µL of RNAse A solution in distilled water and incubate at 37 °C for 1 h.
  2. Use 3 µL of genomic DNA for PCR amplification. Perform PCR screening of the selected clones using primers annealing within the neomycin resistance gene and subtelomere 15q. PCR amplification is performed using standard polymerase enzymes and PCR protocols21.
    NOTE: PCR conditions for MS2 primers (MS2-subtel15q-primer-S and MS2 primer AS) and CTR primers (CTR prime S and CTR primer AS) are the following: 98 °C for 20 s as denaturation step and then 34 cycles at 98 °C 10 s, 58°C 20 s, 72 °C 15 s, using polymerase enzyme in a 25 µL reaction mix (see Table of Materials). Primer sequences are indicated in Table 1.
  3. Run PCR reactions on agarose gel and extract PCR bands obtained from positive clones using standard gel extraction procedures (see Table of Materials for gel extraction reagents).
  4. Perform DNA sequencing analyses of the gel-extracted PCR product for confirmation of the presence of the MS2 sequences21.
    NOTE: The sequencing analyses can be performed using the primers used for the PCR screening.
  5. Southern blot screening of PCR positive clones
    1. Grow the clones positive at PCR and sequencing screening from the original 96 well plate (the backup plate) to 6 well plates in Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), streptomycin (0.2 µg per mL of medium) and containing neomycin at a concentration of 0.7 µg/mL at 37 °C 5% CO2.
      NOTE: Alternatively, if some of these clones have been lost, thaw them from one of the freezing plates (see protocol 2.6).
    2. Once the clones are at 90% confluence in the 6 well plate, wash the cells with PBS and add 250 µL of lysis buffer containing 0.5 µg proteinase K per well.
    3. Scrape the cells using a cell scraper and transfer the lysate in a 1.5 mL tube.
    4. Incubate at 37 °C for 16 h.
    5. Add 1 mL of 100% ethanol, shake vigorously, and allow the DNA to precipitate at least 2 h or overnight at -20 °C.
      NOTE: The protocol can be paused here.
    6. Spin at 13,400 x g at 4 °C for 10 min, discard the supernatant, and wash the pellets with 70% ethanol. Let the pellets air dry at room temperature. Alternatively, use a vacuum concentrator.
    7. Resuspend the DNA pellets in 50 µL of distilled water containing RNAse A and incubate for 1 h at 37 °C.
    8. Digest 5-10 µg of genomic DNA using NcoI and BamHI restriction enzymes (two independent digestions) in 100 µL reaction volume by incubating the digestion reactions at 37 °C overnight.
    9. Run 4 µL of the digestion on agarose gel (0.8% agarose) for complete digestion confirmation.
    10. Add 1/10 volume of sodium acetate 3 M solution pH 5.2 and 2 volumes of 100% ethanol to the restriction digestion reactions and incubate at least 2 h or overnight at -20°C to precipitate DNA.
      NOTE: The protocol can be paused here.
    11. Centrifuge at 13,400 x g at 4 °C for 20 min, discard the supernatants, and wash the pellets with 70% ethanol. Allow the pellets to air dry at room temperature. Alternatively, use a vacuum concentrator.
    12. Resuspend pellets in 20 µL of distilled water and load the digested DNA on a 0.8% agarose gel.
      NOTE: For a better resolution of the digested DNA, prepare a gel at least 15 cm long and run overnight at low voltage ( ̴30 volts). The electrophoresis set up should be optimized.
    13. The following day, stain the gel with a DNA labelling agent, such as ethidium bromide at 1 µL/10 mL final concentration, for 30 min at room temperature and take a picture with a ruler close to the gel on a gel imaging instrument.
    14. Set the transfer of DNA to a nylon membrane and perform membrane hybridization with a MS2 sequence-specific probe using standard procedures21.
    15. Thaw the clones that are positive at PCR, DNA sequencing and Southern blot from one of the two freezing plates (see next step).
  6. Thawing of clones
    1. Prepare one 15 mL tube containing 5 mL of pre-warmed Ham's F-12K (Kaighn's) medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (0.5 units per mL of medium), and streptomycin (0.2 µg per mL of medium) for each clone to be thawed.
    2. Remove one of the freezing plates from -80° and add 100 µL of pre-warmed F12K complete medium to the well containing the positive clone to be thawed.
      NOTE: This procedure should be performed quickly and the 96 well plate should be placed on dry ice after each clone is thawed, in order to allow the other clones to remain frozen. This is particularly important if multiple clones need to be thawed from the same 96 well plate.
    3. Transfer the cells to the 15 mL tube containing 5 mL of medium and centrifuge at 800 x g for 5 min at room temperature.
    4. Aspirate the medium, resuspend the cells in 500 µL of pre-warmed complete F12K medium, and transfer each clone to a single well of a 12 well plate.
  7. Elimination of the neomycin resistance gene from the MS2 cassette integrated at subtelomere 15q.
    1. Allow the clones to grow from a 12 well plate to a 10 cm dish in complete F12K medium.
      NOTE: Neomycin should not be included in the medium unless otherwise indicated.
    2. Add the Cre-expressing adenovirus to the cells cultured in a 10 cm dish at 70% confluence in 10 mL of complete F12K medium.
      NOTE: Using a Cre-GFP expressing adenovirus will allow evaluation of the efficiency of infection, which should approach 100%. The replication-defective adenovirus will be lost after few passages in culture.
    3. 48 hours after infection, split the cells in three 10 cm dishes. Two dishes will be used for verification of the neomycin gene removal by negative selection, growing the cells in neomycin-containing medium (first dish), and by Southern blot (second dish).
    4. Culture the third dish containing the clone for cell freezing and RNA extraction.

3. Verification of TERRA-MS2 Transcript Expression by RT-qPCR

  1. Upon verification of neomycin gene removal, perform total RNA extraction from TERRA-MS2 clones using organic solvents (phenol and guanidine isothiocynate solution)21.
    1. Resuspend the RNA extracted from a 10 cm dish in 100 µL of diethyl pyrocarbonate (DEPC) water.
    2. Run 3 µL of RNA on a denaturating (1% formaldehyde-containing) 1x 3-(N-Morpholino) propanesulfonic acid (MOPS) gel in order to verify concentration and integrity of the RNA. Also analyse RNA concentration using a spectrophotometer.
    3. Treat 3 µg of RNA with DNAse I using 1 unit of DNAse I enzyme in 60 µL final reaction volume.
    4. Incubate the reaction for 1 h at 37 °C.
  2. Reverse transcription reaction and qPCR analyses
    1. Add the following components to a nuclease-free microcentrifuge tube: 2 µL of a 1 µM TERRA specific primer, 1 µL of dNTPs mix (10 mM each), 6 µL of DNAse I-treated RNA (corresponding to ̴300ng RNA). Adjust the volume to 13 µL with 4 µL of DEPC water.
      NOTE: For each RNA to be analysed, a second tube containing the same reagents but a reference specific primer, instead of TERRA specific primer, should be prepared.
    2. Heat the mixture to 65 °C for 5 min and incubate in ice for at least 1 min.
    3. Collect the content of the tubes by brief centrifugation and add 4 µL of 5X RT enzyme Buffer, 1 µL 0.1 M dithiothreitol (DTT), 1 µL (4 units) of RNAse inhibitor, and 1 µL of reverse transcriptase (see Table of Materials).
    4. Incubate the samples at 42 °C for 60 min, then use 2 µL of the RT reaction for qPCR analyses.
  3. Prepare qPCR reaction mix in a final volume of 20 µL consisting of 10 µL of 2x qPCR master mix, 2 µL of cDNA template, 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), and 6 µL of water.
  4. Perform qPCR reaction in a thermocycler using standard protocols21.

4. Production of a MS2-GFP Expressing Retrovirus

  1. To generate MS2-GFP expressing retrovirus, transfect 80% confluent phoenix packaging cells with a MS2-GFP fusion protein expressing retrovirus vector (pBabe-MS2-GFP PURO) and an env gene expressing vector (such as pCMV-VSVG) using a molar ratio 4:1 of the two vectors.
  2. On the following day, replace the culturing medium (DMEM supplemented with 10% FBS, 2 mM L-glutamine and Pen/Strep) with fresh medium containing 10mM sodium butyrate.
  3. Incubate for 8 h at 37 °C, 5% CO2, then replace the medium with fresh culturing medium from which the virus will be collected.
  4. After 48 h, remove the retrovirus-containing medium from the phoenix cells. This medium can be directly used for infection of TERRA-MS2 clones, in which case filter the medium through a 0.45 µm filter, add polybrene (30 µg/mL final concentration) and add it to the cells (in this case the protocol continues at step 5). Alternatively, retrovirus can be precipitated in a 50 mL falcon tube by adding 1/5th volume of 50% PEG-8000/900 mM NaCl solution and incubating overnight at 4 °C on a rotator wheel.
  5. On the following day, pellet retrovirus particles by centrifugation at 2,000 x g for 30 min, remove supernatant, and resuspend the pellet in F12K medium without serum.
    NOTE: The virus particles can be resuspended in 1/100th of the original supernatant volume. An infection test should be performed in order to verify the minimum volume of retrovirus required to efficiently infect the cells.

5. Visualization of TERRA-MS2 Transcripts in Living Cells

  1. Plate TERRA-MS2 clones and WT AGS cells in glass-bottomed dishes. On the day of infection, add polybrene to the medium (30 µg/mL final concentration) and the MS2-GFP expressing retrovirus.
  2. After 24 hours, discard the virus-containing medium and add fresh medium without phenol-red.
  3. Analyze the cells at an inverted microscope using the appropriate microscope setting. Image the cells with a 100X or 60X objective with large numerical aperture (1.4X) and using a sensitive camera (EMCCD). Use an environmental control system to maintain the samples at 37 °C and 5% CO2 during imaging.

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结果

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图 1 展示了实验策略的总体概述。图中显示了在 AGS 细胞中构建 TERRA-MS2 克隆的主要实验步骤及相应的时间安排（图 1A）。第 1 天，将 MS2 插入片段和表达 sgRNA/Cas9 的载体共转染至 6 孔板的多个孔中（如 图 1B 所示）。两条不同的 15q 端粒近端特异性引导 RNA 序列被克隆至表达 Cas9 切口酶的 pX335 载体中，生成两种 sgRNA-pX335 载体，并与 MS2 插入片段共同转染。可将其中一个孔转染表达 GFP 的载体，以验证转染效率。第 2 天，将细胞用胰蛋白酶消化，并从单个孔转移至含筛选培养基的 10 cm 培养皿中。第 3 天更换培养基，此时大多数未转染的细胞已死亡。细胞在筛选培养基中持续培养，直至可见单克隆形成并可用于挑取。在此期间，不应进行胰蛋白...

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讨论

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本文介绍一种生成含MS2序列的人类癌细胞克隆的方法，其中MS2序列已整合至15q亚端粒区域。利用这些克隆，可通过共表达MS2-GFP融合蛋白，利用荧光显微镜检测由15q亚端粒转录的MS2标记的TERRA分子。该方法使研究人员能够在活细胞中研究来自单个端粒的TERRA动态行为21。在本实验方案中，TERRA-MS2克隆选自AGS细胞系，该细胞系是研究TERRA的一个理想模型系统，因为TERRA在人类胃癌样本中的表达水平上调24。然而，本文所述方案可经调整后适用于其他细胞系中TERRA-MS2克隆的筛选。原则上，通过使用特异性的MS2表达盒和CRISPR策略，可将MS2序列整合至除15号染色体端粒以外的其他亚端粒区域。本方法中采用Cas9切口酶（nickase）和双导向RNA，以提高整合的特异性22。采用该策略，在AGS细胞中预计可鉴定出约2%的阳性克隆。也可尝试测试其他类型的Cas9酶，以提高克隆筛选的成功率25。此外，为最大化克隆筛选效率，需特别注...

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披露

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作者声明不存在竞争性财务利益

致谢

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我们感谢特伦托大学CIBIO先进影像中心以及维也纳马克斯·F·佩鲁茨实验室（MFPL）生物光学显微镜设施的工作人员。本研究工作获得了Mahlke-Obermann基金会以及欧盟第七框架计划（研究、技术发展与示范项目）的资助，项目编号为609431，授予EC。EC还获得了意大利教育大学与研究部（MIUR） Rita Levi Montalcini博士后奖学金的支持。

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材料

本文使用的材料清单
姓名公司目录编号评论
AGS细胞--Christian Baron（Université 德蒙特é等）
F12K 营养混合物 1XGIBCO21127022AGS细胞培养基
L-谷氨酰胺 CORNINGMT25005CI细胞培养基成分
青霉素-链霉素溶液CORNING30-002-CI细胞培养基成分
胎牛血清Sigma AldrichF2442细胞培养基成分
DMEM 1XGIBCO21068028phoenix细胞培养基
CaCl2Sigma AldrichC1016用于凤凰细胞转染
HEPESSigma AldrichH3375用于凤凰细胞转染（HBS 溶液）
KClSigma AldrichP9333用于凤凰细胞转染（HBS 溶液）
葡萄糖Sigma AldrichD9434用于凤凰细胞转染（HBS 溶液）
NaClSigma AldrichS7653用于凤凰细胞转染（HBS 溶液）和逆转录病毒沉淀
Na2HPO4Sigma AldrichS3264用于Phoenix细胞转染（HBS溶液）
1×胰蛋白酶EDTA溶液CORNING59430C用于细胞传代
DPBS 1XGIBCO14190250杜氏磷酸盐缓冲液
DMSOSigma AldrichD8418细胞冻存液组分（80% FBB 和 20% DMSO）
G-418 硫酸二氢盐福麦迪亚G4185筛选药物 
明胶溶液 生物试剂Sigma AldrichG139396孔DNA板的封板与冻存
Tris碱Fisher BioReagents10376743基因组DNA提取用细胞裂解缓冲液组分
EDTASigma AldrichE6758基因组DNA提取用细胞裂解缓冲液组分
SDSSigma Aldrich71729基因组DNA提取用细胞裂解缓冲液组分
蛋白酶K赛默飞世尔科技AM2546基因组DNA提取用细胞裂解缓冲液组分
RNA酶A赛默飞世尔科技12091021DNA 提取过程中的 RNA 降解
琼脂糖Sigma AldrichA5304DNA凝胶制备
Atlas ClearSightBioatlasBH40501用于在琼脂糖凝胶中检测DNA和RNA样品的染色试剂
乙醇Fisher BioReagentsBP28184DNA沉淀
乙酸钠 Sigma Aldrich71196用于在3M浓度、pH 5.2条件下进行DNA沉淀
Wizard SV 凝胶与 PCR 纯化系统PromegaA9282从琼脂糖凝胶中提取PCR片段以进行新霉素阳性克隆的PCR筛选
TrizolAMBION15596018用于RNA提取的有机溶剂
Dnase I赛默飞世尔科技89836基因组DNA从RNA中的降解 
dNTPs 混合物Invitrogen10297018用于逆转录和PCR反应
DTTInvitrogen707265ML用于RT反应
焦碳酸二乙酯Sigma AldrichD5758用于以1:1000稀释度使水中的RNase失活
Ribolock赛默飞世尔科技EO0381RNase 抑制剂
MOPSSigma AldrichM9381RNA凝胶的制备
多聚甲醛电子显微镜科学公司15710变性RNA凝胶的制备（含1% PFA的1x MOPS）
Superscript III 逆转录酶Invitrogen18080-093逆转录反应
Pfu DNA聚合酶（重组）赛默飞世尔科技EP0501PCR反应
2X qPCRBIO SyGreen Mix Separate-ROXPCR BIOSYSTEMSPB 20.14qPCR 反应
Cre-GFP 腺病毒https://medicine.uiowa.edu/vectorcore1174-HT用于感染 TERRA-MS2 克隆以去除 neomycn 基因
丁酸钠Sigma AldrichB5887用于促进Phoenix细胞中逆转录病毒颗粒的产生
PEG8000Sigma Aldrich89510逆转录病毒颗粒的沉淀
35µ玻璃底培养皿Ibidi81158用于TERRA-MS2克隆的活细胞成像分析

参考文献

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TERRA MS2 GFP RNA FISH TRF2 mCherry

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