搜索

A Simple Method to Generate Gene Knockout Clones in Human Cells Using Transcription Activator-Like Effector Nuclease (TALEN)
采用转录激活因子样效应物核酸酶(TALEN)在人细胞中产生基因敲除克隆的简单方法

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Transcription activator-like effectors (TALEs) are naturally occurring proteins secreted by the plant pathogen, Xanthomonas, and fused to the Fok1 endonuclease to generate TALE nucleases (TALENs). TALEN pairs bind to specific DNA sequences initiating Fok1 dimerization and double-stand cleavage of DNA within the TALEN target site. This cleavage event triggers cellular repair mechanisms that result in insertions and/or deletions (indels), which enable gene knockout. The high specificity and efficiency of TALENs makes them important tools for genome editing. Here, we describe a method for the generation of single-cell clones with targeted gene knockout by TALEN using co-transfection and FACS with a fluorescent reporter. This protocol was designed to knockout cell death-inducing DFFA-like effector b, CIDEB, in Huh7.5 cells; however, this protocol can be applied to a wide range of cell types and genes of interest.

Materials and Reagents

  1. Huh7.5 cells (Charles Rice lab, Rockefeller University)
  2. Dulbecco’s modified Eagle’s medium (DMEM high glucose) (HyClone, catalog number: SH30243.02 )
  3. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10437-028 )
  4. Antibiotic Antimycotic solution (10,000 U/ml penicillin G, 10,000 µg/ml streptomycin, 25 µg/ml amphotericin B) (HyClone, catalog number: SV30079.01 )
  5. MEM nonessential amino acids (NEAA) (Corning Incorporated, catalog number: 25025CI )
  6. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: BP39920 )
  7. Trypsin, 0.05% (HyClone, catalog number: SH30236.02 )
  8. TALEN Sure KO plasmids (Cellectis)
    Note: It is custom designed to target the gene of interest and  includes right and left TALEN plasmids to bind both sequences of DNA flanking the target site.
  9. pcDNA3-EGFP expression plasmid (Addgene, plasmid number: 13031 )
  10. Lipofectamine 2000 Transfection Reagent (Life Technologies, InvitrogenTM, catalog number: 11668019 )
  11. Opti-MEM I Reduced-Serum Medium (Life Technologies, Gibco®, catalog number: 31985070 )
  12. QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE09050 )
  13. Platinum PCR SuperMix High Fidelity (Life Technologies, InvitrogenTM, catalog number: 12532016 )
  14. Primers flanking the TALEN target site (custom DNA nucleotides) (Integrated DNA Technologies)
  15. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  16. Tris-HCl buffer (pH 6.8, 0.5 M) (Bio-Rad Laboratories, AbD Serotec®, catalog number: 1610799 )
  17. Sodium dodecyl sulfate (20% solution) (Thermo Fisher Scientific, catalog number: BP13111 )
  18. Glycerol (Fisher Scientific, catalog number: BP2291 )
  19. Bromophenol blue (Thermo Fisher Scientific, catalog number: B3925 )
  20. 2-mercaptoethanol (Thermo Fisher Scientific, catalog number: O3446I100 )
  21. 10% DMEM culture media (see Recipes)
  22. 20% DMEM culture media (see Recipes)
  23. FACS buffer (see Recipes)
  24. 2x SDS loading buffer (see Recipes)

Equipment

  1. Cell culture incubator at 37 °C and 5% CO2
  2. Cell culture-treated polystyrene plates (100 mm, 6 well, 12 well, 24 well, 48 well, 96 well) (Corning Incorporated)
  3. Polystyrene Round-Bottom Tube (5 ml FACS tubes) (BD Biosciences, Falcon®, catalog number: 352008 )
  4. Round-Bottom Tube Cap (5 ml) (BD Biosciences, Falcon®, catalog number: 352032 )
  5. 40 micron cell strainer (Falcon®, catalog number: 352340 )
  6. Fluorescence microscope
  7. Table top centrifuge
  8. FACSAria cell sorter (BD Biosciences, catalog number: 656700 )
  9. Multi-channel pipette
  10. Western blotting equipment (Bio-Rad Laboratories, AbD Serotec®)
  11. Heat block (VWR international)
  12. PCR thermocycler (Bio-Rad Laboratories, AbD Serotec®)
  13. Agarose gel electrophoresis system
  14. 3730 DNA analyzer (Life Technologies, Applied Biosystems®, catalog number: 3730S )

Procedure

  1. Co-transfection of TALEN and EGFP plasmids into Huh7.5 cells
    1. Plate Huh7.5 cells onto 100 mm cell culture plates in 15 ml 10% DMEM culture media without antibiotics 18-24 h before transfection such that the cells will be ~60-70% confluent at the time of transfection. Scale up and down the culture according to the manufacturer’s protocol for the cell type used.
    2. Combine 8 µg of the right TALEN, 8 µg of the left TALEN and 8 µg of EGFP plasmid in 1.5 ml Opti-MEM medium. Mix gently. Prepare separate tubes for controls, such as mock transfection, which controls for potential side effects of the transfection reagent (only to be included in step A).
    3. In a separate tube, add 60 µl Lipofectamine 2000 to 1.5 ml Opti-MEM medium for each sample. Mix gently and incubate at room temperature for 5 min.
    4. Add the DNA-Opti-MEM solution (from step A2) to the Lipofectamine-Opti-MEM solution (from step A3). Thoroughly mix the solution by gently pipetting up and down. Incubate at room temperature for 20 min.
    5. Add 1.5 ml of the DNA-lipid complex to the cells drop-wise and gently rock the plate back and forth to evenly distribute the complexes.
    6. (Optional) ~6 h later, replace the media with fresh 10% DMEM culture media.
    7. Incubate the cells at 37 °C with 5% CO2 for 48 h.
    8. Approximate transfection efficiency by measuring the number of GFP positive cells using a fluorescence microscope.

  2. Enriching for GFP positive cells using fluorescence activated cell sorting (FACS)
    1. 48 h post-transfection, wash cells with 1x PBS, then trypsinize cells and pipette up and down forcefully to break up cell aggregates.
    2. Add 10% DMEM culture media to inactivate trypsin.
    3. Centrifuge cells at 200 x g for 10 min.
    4. Aspirate supernatant and resuspend the cell pellet in FACS buffer (see Recipes) at a concentration of 10 x 106 cells/ml. Pipette up and down thoroughly to attain single cell suspensions.
    5. Place a cell strainer over a FACS tube (sterilized in ethanol) and carefully pipet the cells through the filter into the tube to remove cell clumps. Cells should be sorted immediately or placed on ice and covered until ready to be sorted.
    6. Sort the GFP positive cells into 20% DMEM culture media using the FACSAria cell sorter.
    7. Cell viability is typically diminished following transfection and sorting. Thus, the sorted cells should be seeded at a fairly high cell density in fresh 20% DMEM culture media and allowed to recover for ~1 week. Replace the media every 3-4 days during this recovery time.
    Note: If cells fail to recover, repeat the experiment starting with a larger cell number.

  3. Generation of single-cell clones by serial dilution onto 96 well plates
    1. Once the cells have recovered, add 100 µl of 20% DMEM culture media to all but the first well, A1, of a 96 well plate.
    2. Suspend sorted cells at a concentration of 1 x 104 cells/ml.
    3. Add 200 µl of sorted cell suspension to well A1, then transfer 100 µl to well B1, and so on, until all the wells in the first column contain cells. Discard 100 µl from H1, and then add 100 µl of media to wells A1-H1 for a final volume of 200 µl.
    4. Using a multi-channel pipette, transfer 100 µl from the first column to the second column and repeat for all columns. Bring the final volume of all wells to 200 µl.
    5.  Incubate cells at 37 °C with 5% CO2 in 20% DMEM culture media. Analyze the wells daily marking those that contain a single cell. Close monitoring and stringency is crucial at this step for obtaining single-cell clones.
    6. Allow single-cell clones to become confluent in 96 well plates, then gently trypsinize and transfer to 48 well plates. Repeat this process so that the cells are slowly expanded to larger surface areas to promote cell growth (e.g. transfer from 96 well to 48 well to 24 well to 12 well to 6 well plates).

  4. Analyze protein expression by Western blotting to identify single-cell clones with gene knockout
    1. In 12 well plates, wash cells with 1x PBS then collect cells by trypsinization. Add 10% DMEM culture media to inactivate trypsin.
    2. Centrifuge cells at 200 x g for 10 min.
    3. Aspirate supernatant and lyse the cells with 2x SDS loading buffer (see Recipes) to extract protein.
    4. Immediately boil samples at 100 °C for 10 min using a hotplate, then centrifuge at max speed for 1 min. Samples can be stored at -20 °C.
    5. Analyze protein expression via Western blotting (Figure 1A).

  5. Confirm gene knockout by DNA sequence analysis
    1. Extract DNA using QuickExtract DNA solution per the manufacturer’s protocol.
    2. Perform PCR with genomic primers flanking the TALEN target site.
    3. Analyze the PCR products by gel electrophoresis.
    4. Isolate and purify DNA using the Qiagen gel extraction kit per the manufacturer’s protocol.
    5. Sequence DNA to confirm the presence of TALEN-induced indels that result in the loss of gene expression (Figure 1B).

Representative data



Figure 1. Representative data. A. Representative Western blot for the identification of 4 clones negative for CIDEB protein expression, suggesting successful gene knockout by TALEN. B. Representative DNA sequence analysis of clone 3 confirming that the target region contains TALEN-induced indels leading to loss of gene expression.

Notes

  1. Pertaining to step B, Huh7.5 cells display low transfection efficiencies (~10-15%); thus, FACS is an important step to enrich for GFP+ cells. However, sorting may not be necessary for cell lines with high transfection efficiencies such as 293T, HeLa, and RKO cells. If the transfection efficiency is determined to be over 80% by fluorescence microscopy and/ or flow cytometry, then the transfected cells from step A may be directly used for single-cell cloning in step C.
  2. Various protocols exist for single-cell cloning including by serial dilution (described here), by cloning rings, and by directly counting and plating cells at 0.5 cells per well. The best method will be determined by the cell line used and viability of the cells after gene knockout.
  3. Depending on the available equipment and size of the screen, it may be easier to analyze the clones at the DNA level first, and then confirm positive clones by Western blotting. This is at the experimenter’s discretion.

Recipes

  1. 10% DMEM culture media
    DMEM supplemented with 10% FBS, 1x NEAA, 100 U/ml penicillin G, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B
  2. 20% DMEM culture media
    DMEM supplemented with 20% FBS, 1x NEAA, 100 U/ml penicillin G, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B
  3. FACS buffer
    PBS with 5% FBS
  4. 2x SDS gel-loading buffer
    0.1 M Tris-HCl (pH 6.8) with 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.2% bromophenol blue, and 10% β-mercaptoethanol

Acknowledgments

This work was supported by NIH grant R01 A1079150.

References

  1. Wu, X., Lee, E. M., Hammack, C., Robotham, J. M., Basu, M., Lang, J., Brinton, M. A. and Tang, H. (2014). Cell death-inducing DFFA-like effector b is required for hepatitis C virus entry into hepatocytes. J Virol 88(15): 8433-8444.

简介

转录激活子样效应器(TALE)是由植物病原体黄单胞菌分泌的天然存在的蛋白质,并且与Fok1内切核酸酶融合以产生TALE核酸酶(TALEN)。 TALEN对与特异性DNA序列结合,启动FAL1二聚化和在TALEN靶位点内双链切割DNA。 该切割事件触发导致插入和/或缺失(插入缺失)的细胞修复机制,其使得能够进行基因敲除。 TALEN的高特异性和高效性使其成为基因组编辑的重要工具。 在这里,我们描述了使用共转染和FACS与荧光报告基因通过TALEN产生具有靶向基因敲除的单细胞克隆的方法。 该方案设计为在Huh7.5细胞中敲除诱导细胞死亡的DFFA样效应子b,CIDEB; 然而,该协议可以应用于广泛的细胞类型和感兴趣的基因。

材料和试剂

  1. Huh7.5细胞(Charles Rice lab,Rockefeller University)
  2. Dulbecco改良的Eagle培养基(DMEM高葡萄糖)(HyClone,目录号:SH30243.02)
  3. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10437-028)
  4. 抗生素抗真菌溶液(10,000U/ml青霉素G,10,000μg/ml链霉素,25μg/ml两性霉素B)(HyClone,目录号:SV30079.01)
  5. MEM非必需氨基酸(NEAA)(Corning Incorporated,目录号:25025CI)
  6. 材料和试剂

    1. Huh7.5细胞(Charles Rice lab,Rockefeller University)
    2. Dulbecco改良的Eagle培养基(DMEM高葡萄糖)(HyClone,目录号:SH30243.02)
    3. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10437-028)
    4. 抗生素抗真菌溶液(10,000U/ml青霉素G,10,000μg/ml链霉素,25μg/ml两性霉素B)(HyClone,目录号:SV30079.01)
    5. MEM非必需氨基酸(NEAA)(Corning Incorporated,目录号:25025CI)
    6. ...
    7. QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE09050)
    8. Platinum PCR SuperMix High Fidelity (Life Technologies, InvitrogenTM, catalog number: 12532016)
    9. Primers flanking the TALEN target site (custom DNA nucleotides) (Integrated DNA Technologies)
    10. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704)
    11. Tris-HCl buffer (pH 6.8, 0.5 M) (Bio-Rad Laboratories, AbD Serotec®, catalog number: 1610799)
    12. Sodium dodecyl sulfate (20% solution) (Thermo Fisher Scientific, catalog number: BP13111)
    13. 甘油(Fisher Scientific,目录号:BP2291)
    14. 溴酚蓝(Thermo Fisher Scientific,目录号:B3925)
    15. 2-巯基乙醇(Thermo Fisher Scientific,目录号:O3446I100)
    16. 10%DMEM培养基(参见配方)
    17. 20%DMEM培养基(参见配方)
    18. FACS缓冲区(参见配方)
    19. 2x SDS加样缓冲液(见配方)

    设备

    1. 在37℃和5%CO 2/s的细胞培养箱中培养
    2. 细胞培养处理的聚苯乙烯板(100mm,6孔,12孔,24孔,48孔,96孔)(康宁公司)
    3. 聚苯乙烯圆底管(5ml FACS管)(BD Biosciences,Falcon ,目录号:352008)
    4. 圆底管帽(5ml)(BD Biosciences,Falcon ,目录号:352032)
    5. 40微米细胞过滤器(Falcon ,目录号:352340)
    6. 荧光显微镜
    7. 台式离心机
    8. FACSAria细胞分选仪(BD Biosciences,目录号:656700)
    9. 多通道移液器
    10. Western印迹设备(Bio-Rad Laboratories,AbD Serotec )
    11. 热块(VWR国际)
    12. PCR热循环仪(Bio-Rad Laboratories,AbD Serotec )
    13. 琼脂糖凝胶电泳系统
    14. 3730 DNA分析仪(Life Technologies,Applied Biosystems ,目录号:3730S)

    程序

    1. 将TALEN和EGFP质粒共转染到Huh7.5细胞中
      1. 板Huh7.5细胞在100毫米细胞培养板上在15毫升10%DMEM 培养基中不含抗生素18-24 h,转染前使用 在转染时细胞将〜60-70%汇合。 规模 根据制造商的方案对培养物进行上下培养   使用的细胞类型。
      2. 结合8μg的右TALEN,8μg的 左TALEN和8μgEGFP质粒在1.5ml Opti-MEM培养基中。 混合 轻轻地。 准备控制单独的管,如模拟转染, 其控制转染试剂的潜在副作用 (仅包括在步骤A中)。
      3. 在单独的管中,加入60μl Lipofectamine 2000至1.5ml Opti-MEM培养基。 轻轻混合   并在室温下孵育5分钟
      4. 加入DNA-Opti-MEM 溶液(来自步骤A2)至Lipofectamine-Opti-MEM溶液 步骤A3)。 通过轻轻地吸取上下溶液彻底混合溶液。 在室温下孵育20分钟。
      5. 加入1.5 ml的 DNA-脂质复合物到细胞中,轻轻地摇动平板 并均匀分布复合物。
      6. (可选)〜6 h后,更换新鲜的10%DMEM培养基的培养基。
      7. 在37℃下用5%CO 2孵育细胞48小时。
      8. 通过使用荧光显微镜测量GFP阳性细胞的数量,近似转染效率
    2. 使用荧光激活细胞分选(FACS)富集GFP阳性细胞
      1. 转染后48小时,用1x PBS洗涤细胞,然后胰蛋白酶化细胞   并向上和向下移液以强力打碎细胞聚集体
      2. 加入10%DMEM培养基以灭活胰蛋白酶。
      3. 在200×g离心细胞10分钟。
      4. 吸出上清液,并在FACS缓冲液中重悬细胞沉淀 Recipes),浓度为10×10 6个细胞/ml。 移液器上下移动 彻底获得单细胞悬浮液。
      5. 放置单元格 过滤器通过FACS管(在乙醇中灭菌)和小心吸取 细胞通过过滤器进入管中以除去细胞团块。 细胞 应立即分类或放在冰上覆盖,直到准备好  
      6. 使用FACSAria细胞分选仪将GFP阳性细胞分选到20%DMEM培养基中。
      7. 细胞活力通常在转染后减少 排序。 因此,分选的细胞应该在相当高的细胞接种 密度在新鲜的20%DMEM培养基中,并允许恢复〜1 周。 在此恢复时间内每3-4天更换一次介质。
      注意:如果单元格无法恢复,请从更大的单元格编号开始重复实验。

    3. 通过在96孔板上连续稀释产生单细胞克隆
      1. 一旦细胞已恢复,添加100微升20%DMEM培养基到96孔板的第一个孔,除了A1。
      2. 悬浮分选的细胞,浓度为1×10 4个细胞/ml。
      3. 加入200微升的分选细胞悬液到孔A1,然后转移100微升 到井B1,依此类推,直到第一列中的所有井都包含 细胞。 从H1中弃去100μl,然后向孔中加入100μl培养基 A1-H1,最终体积为200μl。
      4. 使用多通道 移液器,从第一列转移100微升到第二列和 对所有列重复。 使所有孔的最终体积为200μl
      5. &在37℃下用20%DMEM培养基中的5%CO 2孵育细胞。 分析   每天井标记那些包含单个细胞。 关 监测和严格性是获得这一步骤的关键 单细胞克隆。
      6. 允许单细胞克隆汇合 在96孔板中,然后轻轻地胰蛋白酶消化并转移至48孔 板。 重复这个过程,使细胞缓慢扩展到 更大的表面积以促进细胞生长(例如从96孔转移) 至48孔至24孔至12孔至6孔板)。

    4. 通过蛋白质印迹分析蛋白质表达以鉴定具有基因敲除的单细胞克隆
      1. 在12孔板中,用1×PBS洗涤细胞,然后收集细胞 胰蛋白酶消化。 加入10%DMEM培养基以灭活胰蛋白酶
      2. 以200×g离心细胞10分钟。
      3. 吸出上清液并用2x SDS上样缓冲液(见Recipes)裂解细胞以提取蛋白质
      4. 立即使用热板在100℃下煮沸样品10分钟,然后 以最大速度离心1分钟。 样品可以储存在-20°C。
      5. 通过蛋白质印迹分析蛋白质表达(图1A)
    5. 通过DNA序列分析确认基因敲除
      1. 使用QuickExtract DNA溶液根据制造商的协议提取DNA。
      2. 用位于TALEN靶位点侧翼的基因组引物进行PCR。
      3. 通过凝胶电泳分析PCR产物
      4. 使用Qiagen凝胶提取试剂盒根据制造商的方案分离和纯化DNA
      5. 序列DNA以确认导致基因表达丢失的TALEN诱导的indel的存在(图1B)。

    代表数据



    图1。 代表性数据 。 A.用于鉴定CIDEB蛋白表达阴性的4个克隆的代表性Western印迹,表明TALEN基因敲除 。 B.克隆3的代表性DNA序列分析,证实靶区域含有导致基因表达丧失的TALEN诱导的缺口。

    笔记

    1. 对于步骤B,Huh7.5细胞显示低转染效率(〜10-15%);因此,FACS是富集GFP +细胞的重要步骤。然而,对于具有高转染效率的细胞系如293T,HeLa和RKO细胞,分选可能不是必需的。如果通过荧光显微镜和/或流式细胞术确定转染效率超过80%,则来自步骤A的转染细胞可以直接用于步骤C中的单细胞克隆。
    2. 存在用于单细胞克隆的各种方案,包括通过连续稀释(如本文所述),通过克隆环,并通过直接计数并以0.5细胞/孔接种细胞。最佳方法将由所使用的细胞系和基因敲除后细胞的存活力确定。
    3. 根据可用的设备和筛选的大小,可以更容易地首先在DNA水平分析克隆,然后通过Western印迹确认阳性克隆。这是由实验者自行决定。

    食谱

    1. 10%DMEM培养基
      补充有10%FBS,1×NEAA,100U/ml青霉素G,100μg/ml链霉素和0.25μg/ml两性霉素B的DMEM
    2. 20%DMEM培养基
      补充有20%FBS,1×NEAA,100U/ml青霉素G,100μg/ml链霉素和0.25μg/ml两性霉素B的DMEM
    3. FACS缓冲区
      含5%FBS的PBS
    4. 2x SDS凝胶上样缓冲液
      含有4%十二烷基硫酸钠(SDS),20%甘油,0.2%溴酚蓝和10%β-巯基乙醇的0.1M Tris-HCl(pH6.8)

    致谢

    这项工作是由NIH授权R01 A1079150支持。

    参考文献

    1. Wu,X.,Lee,E. M.,Hammack,C.,Robotham,J. M.,Basu,M.,Lang,J.,Brinton,M.A。和Tang,H。 细胞死亡诱导性DFFA样效应物b是丙型肝炎病毒进入肝细胞所必需的。 a> J Virol 88(15):8433-8444。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Hammack, C. (2015). A Simple Method to Generate Gene Knockout Clones in Human Cells Using Transcription Activator-Like Effector Nuclease (TALEN). Bio-protocol 5(14): e1531. DOI: 10.21769/BioProtoc.1531.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。