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Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells
在果蝇细胞中利用RNAi联合基于CRISPR的基因敲除技术进行协同致死筛选   

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Abstract

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).

Keywords: Synthetic lethality(协同致死), Screening(筛选), RNAi(RNAi), Drosophila(果蝇), S2R+ cells(S2R +细胞), Cell culture(细胞培养), CRISPR(CRISPR)

Background

Knowledge of genetic interactions such as synthetic lethality can be invaluable for determining the functional relationships between genes. For example, large scale genetic interaction screens in yeast were recently used to assemble a global ‘wiring diagram of cellular function’ (Costanzo et al., 2016). Alternatively, specific types of genetic interaction such as synthetic lethal interactions can be used to identify drug targets for diseases including cancer (Kaelin, 2005).

Identification of synthetic interactions requires combinatorial disruption of two genes. A previous method to achieve this in Drosophila cell culture was to deliver multiple dsRNA reagents simultaneously (e.g., Fisher et al., 2015). However, RNAi reagents have limitations including off-target effects and incomplete target knockdown, which are compounded when multiple reagents are delivered together. By combining CRISPR mutagenesis with single dsRNA treatments, these issues are avoided, leading to simpler interpretation of screen results and robust identification of ‘hits’.

Materials and Reagents

  1. 10 cm dish (e.g., Corning, catalog number: 430167 )
  2. T75 flasks (Corning, catalog number: 430641U )
  3. 0.2 μm filter (Thermo Fisher Scientific, catalog number: 156-4020 )
  4. 6-well tissue culture plates (Corning, catalog number: 3516 )
  5. 96-well clear bottom tissue culture plates (Corning, catalog number: 3610 )
  6. 40 μm cell strainer (Corning, Falcon®, catalog number: 352340 )
  7. Parafilm (Parafilm, catalog number: PM-999 )
  8. 24-well plate
  9. 15 ml conical tube
  10. Tips  
  11. S2R+ cells (Drosophila Genomics Resource Center, catalog number: 150 )
  12. sgRNA expression plasmid (pl18 - available from author on request)
  13. act-GFP plasmid (available from author on request)
  14. Chemically competent E. coli cells (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: C404003 )
  15. Effectene Transfection Reagent Kit (QIAGEN, catalog number: 301427 )
  16. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 ) with 1% FBS (GE Healthcare, HyCloneTM, catalog number: SH30541.03 )
  17. HRMA reagents (Housden and Perrimon, 2016)
  18. Zero-Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 450245 )
  19. M13F and M13R primers
  20. dsRNA library in 384-well opaque, white tissue culture plates with 5 μl dsRNA at 50 ng/μl per well (see the Drosophila RNAi Screening Center [http://fgr.hms.harvard.edu/fly-cell-rnai-libraries] for available libraries). Note that this protocol focuses on dsRNA libraries available from the DRSC but additional libraries are also available from other sources.
  21. CellTiter-Glo reagent (Promega, catalog number: G7570 )
  22. Schneider’s Drosophila media (Thermo Fisher Scientific, GibcoTM, catalog number: 21720 )
  23. Penicillin/streptomycin (Thermo Fisher Scientific, Invitrogen, catalog number: 15070063 )
  24. Complete media (10% FBS) (see Recipes)
  25. High-serum media (20% FBS) (see Recipes)
  26. Serum-free Schneider’s media (see Recipes)

Equipment

  1. 10 ml pipette
  2. Humidity chamber (a tupperware box lined with damp paper towels is fine)
  3. 25 °C cell culture incubator (any brand – CO2 regulation is not necessary)
  4. Microscope (e.g., Leica Microsystems, model: DMIL , with 10x objective and any brand of camera)
  5. 12-channel 100 μl pipette (e.g., Thermo Labsystems)
  6. 12-channel 10-50 μl pipette (e.g., Thermo Labsystems)
  7. Centrifuge with plate-compatible rotor (any brand)
  8. Pasteur pipette
  9. Haemocytometer (any brand)
  10. Multichannel reservoir
  11. Timer
  12. Rotator
  13. Plate reader with luminescence reading capability (e.g., Molecular Devices, model: SpectraMax Paradigm )
  14. Fluorescence-activated cell sorter (e.g., BD FACSAria)
  15. Thermal cycler (any brand)

Procedure

  1. Preparation of conditioned media
    1. Grow S2R+ cells to 100% confluency in a 10 cm dish or T75 flask in complete media. Cells should be cultured under standard conditions: 25 °C, with ambient CO2 levels.
    2. Split cells 1:5 using complete media and culture for 3 days (or until almost 100% confluent but without overlapping cells).
    3. Remove all media and replace with 10 ml complete media.
    4. Immediately detach cells by gently pipetting the complete media using a 10 ml pipette to wash the cells off the bottom of the dish/flask, then transfer 5 ml of cell suspension to a new dish or flask and add an additional 5 ml complete media.
    5. Grow for 16 h (no longer). Check to ensure that the cells look healthy at this point.
    6. Collect media and sterilize using a 0.2 μm filter to remove all cells.
    7. Dilute media 1:1 using high-serum media. Store at 4 °C up to four weeks.

  2. Generation of mutant cell lines
    1. Design and generate a sgRNA expression plasmid using the pl18 vector as described previously (Housden et al., 2016).
    2. Seed S2R+ cells in complete media into a 6-well plate such that cells will be 40-80% confluent upon attachment (2-8 x 105 cells per well in 1 ml complete media). Allow to attach for ~20 min at 25 °C.
    3. Transfect cells with 40 ng act-GFP plasmid and 360 ng of sgRNA expression plasmid from step B1 using 10 μl Effectene reagent according to the standard Effectene Transfection Reagent protocol.
    4. Incubate cells at 25 °C for 4 days.
    5. Place 100 μl conditioned media in each well of three clear-bottom 96-well tissue culture plates.
    6. Aspirate spent media from transfected cells and detach by gentle pipetting with 1 ml PBS/1% FBS. Filter cell suspension through a 40 μm filter to remove cell aggregates.
    7. Use FACS to isolate the top 10% of GFP expressing cells, excluding the top 1% of GFP cells (which are generally not viable). See Note 1 and Figure 1.
    8. Seed 100 cells into well A1 of each 96-well culture plate (this will be used to find the focal plane in step B10) and a single cell into the remaining 95 wells of each plate.
    9. Seal plate edges with Parafilm and place in a humidity chamber inside a 25 °C incubator.
    10. Incubate plates for 2 weeks and then examine each well under a microscope to identify those containing colonies, using well A1 to focus the microscope.
    11. For wells containing colonies that have grown to a sufficient size (see Figure 2 for examples of suitable colonies), resuspend the colony in the media already in the well by gentle pipetting with a 100 μl pipette. Mix 50 μl of the cell suspension with 75 μl conditioned media and transfer to a well of a new 96-well plate. Use the remaining 50 μl cell suspension for a genomic DNA preparation for analysis of mutations.
    12. Identify mutant colonies using the previously described genomic DNA and HRMA protocols (Housden and Perrimon, 2016).
    13. Confirm that mutations are homozygous frameshifts by sequencing TOPO-cloned PCR product from step B12 using M13F and M13R primers. We recommend sequencing at least 20 TOPO-cloned constructs for each sample to ensure that all allele sequences of the target gene are identified.
    14. Following identification of mutant clones, the relevant cell population can be expanded by serially transferring from the 96-well plate to a 24-well plate and then a 6-well plate and finally a 10 cm dish or T75 flask. Cells should be transferred using conditioned media until they reach a 6-well plate, at which point complete media can be used. Transfers should be performed when cells reach confluence at each stage.


      Figure 1. FACS setup for isolation of single GFP positive cells. GFP positive cells are isolated by first selecting viable cells (A), then removing doublets (B), then identifying all GFP positive cells (C) and finally selecting the top 10% of GFP expressing cells for sorting (D). Gating hierarchy is shown in panel E.


      Figure 2. Examples of suitable colonies for expansion and sequence analysis

  3. dsRNA screen
    1. Defrost the 384-well dsRNA assay plates from -80 °C storage and centrifuge at 805 x g for 2 min before use.
    2. Remove media from cells cultured in T75 flasks or 10 cm dishes using a Pasteur pipette while taking care not to detach the cells. Note that S2R+ cells are adherent but will detach easily when disturbed.
    3. Add 10 ml serum-free media warmed to room temperature to each T75 flask or 10 cm dish. Use this media to detach the cells by gentle pipetting and transfer to a 15 ml conical tube.
    4. Count cells using a haemocytometer and dilute to 500,000 cells per ml with fresh serum-free media.
    5. Remove seals from each assay plate, holding the plate down while the seal is pulled away.
    6. Transfer diluted cell suspension to a multichannel reservoir and dispense 10 μl (5,000 cells) into each well of each 384-well dsRNA assay plate. Tips do not need to be changed between each well but cell suspension should be pipetted onto the well walls to avoid transferring dsRNA reagents between wells. Start a timer when the first cells are added to the first plate.
    7. Centrifuge plates briefly (130 x g for 5 sec) to move the cell suspension to the bottom of the wells, then place in a sterile hood at room temperature to incubate.
    8. At 45 min after adding the first set of cells, begin to add 35 μl complete media (10% FBS) to each well. Add the complete media to the plates in the same order as the cells were previously added.
    9. Seal plate edges with Parafilm and place into a humidity chamber.
    10. Incubate at 25 °C for 5 days.
    11. Add 30 μl of CellTiter-Glo reagent to each well of the assay plates and gently mix CellTiter-Glo reagent with cells (by placing on a rotator or with gentle shaking for 5 min at room temperature) to allow cells to lyse. Take care not to mix too vigorously to avoid splashing of samples between wells or creation of bubbles.
    12. Read luminescence using a suitable plate reader.

Data analysis

  1. For each plate, remove data for all edge wells (see Note 2) and divide the recorded luminescence values by the median of the respective column, then further divide the new values by the median of each respective row. These transformations account for human error in pipetting and normal variance. See Figure 3 for an illustration of the normalization strategy.


    Figure 3. Data normalization by columns and rows for 36-well example plate. The median of each column is first calculated (A), and each value is then divided by the median of its column (B). The median of each row of transformed data is then calculated (C), and each transformed value is divided by the median of its row (D). The formula for each calculation is shown for the data point in the box.

  2. Convert plate data layout into a single column (a tool for this purpose is available at http://www.flyrnai.org/cgi-bin/DRSC_serialize.pl). Convert all values to log2 to normalize distributions.
  3. Calculate the mean and standard deviation of negative control dsRNA reagents (known non-lethal gene targets and non-targeting dsRNAs – see Note 2 and Figure 4 for details of example negative control locations) for each replicate of the screen in each cell line to identify dsRNAs with viability effects above assay noise (see Notes 3 and 4).
  4. Calculate Z-scores for all data points using mean and standard deviation of controls as calculated in Data analysis step 3 for the respective replicate and cell line: Z = (X-mean)/standard deviation. See http://www.statisticshowto.com/probability-and-statistics/z-score/ for a more detailed description of Z-scores and their interpretation.
  5. Identify hits as dsRNA reagents that reduce viability (Z ≤ -1.5) of at least ⅔ replicates in the mutant cell line and have no viability effect (Z > -1.5) in at least ⅔ replicates in the wildtype cells.

Notes

  1. We recommend performing the FACS isolation of individual cells with an experienced FACS technician. In addition, untransfected cells should be used to determine autofluorescence levels before gating to isolate GFP positive cells.
  2. Edge wells are wells that contain water instead of dsRNA reagents around the edge of each assay plate. These are included because edge wells tend to have higher variability than central wells. See Drosophila RNAi Screening Center (http://fgr.hms.harvard.edu/plate-layouts) for details of typical assay plate layouts. Layout for a test plate is depicted in Figure 4 as an example.


    Figure 4. Example of a plate layout for a Drosophila RNAi Screening Center test plate. Layout of wells varies between libraries. GFP and LacZ do not affect viability of S2R+ cells, and Rho1 and Thread are lethal. Image provided by the DRSC.

  3. We recommend performing three replicate screens. However, given the high costs associated with large scale screens, two replicates can be performed to save time and resources. In this case, additional validation of results may be necessary to identify false positive results.
  4. Z-scores can be calculated using only negative control samples or the entire dataset. For small screens (less than 1,000 genes), we recommend using only negative controls because true hits can have a significant effect on the standard deviation of the whole dataset and therefore reduce sensitivity of hit calling. For large screens, such as with a genome-wide library, calculating Z-scores using the whole dataset provides a better representation of noise within the data.

Recipes

  1. Complete media
    Schneider’s Drosophila media
    10% FBS
    1% penicillin/streptomycin
  2. High serum media
    Schneider’s Drosophila media
    20% FBS
    1% penicillin/streptomycin
  3. Serum-free Schneider’s media
    Schneider’s Drosophila media
    1% penicillin/streptomycin

Acknowledgments

RNAi protocols used here are modified from those developed by the Drosophila RNAi Screening Center (DRSC) (fgr.hms.harvard.edu). Work in the Perrimon lab is supported by the NIH and the Howard Hughes Medical Institute.

References

  1. Costanzo, M., VanderSluis, B., Koch, E. N., Baryshnikova, A., Pons, C., Tan, G., Wang, W., Usaj, M., Hanchard, J., Lee, S. D., Pelechano, V., Styles, E. B., Billmann, M., van Leeuwen, J., van Dyk, N., Lin, Z. Y., Kuzmin, E., Nelson, J., Piotrowski, J. S., Srikumar, T., Bahr, S., Chen, Y., Deshpande, R., Kurat, C. F., Li, S. C., Li, Z., Usaj, M. M., Okada, H., Pascoe, N., San Luis, B. J., Sharifpoor, S., Shuteriqi, E., Simpkins, S. W., Snider, J., Suresh, H. G., Tan, Y., Zhu, H., Malod-Dognin, N., Janjic, V., Przulj, N., Troyanskaya, O. G., Stagljar, I., Xia, T., Ohya, Y., Gingras, A. C., Raught, B., Boutros, M., Steinmetz, L. M., Moore, C. L., Rosebrock, A. P., Caudy, A. A., Myers, C. L., Andrews, B. and Boone, C. (2016). A global genetic interaction network maps a wiring diagram of cellular function. Science 353(6306).
  2. Fischer, B., Sandmann, T., Horn, T., Billmann, M., Chaudhary, V., Huber, W. and Boutros, M. (2015). A map of directional genetic interactions in a metazoan cell. Elife 4.
  3. Housden, B. E., Hu, Y. and Perrimon, N. (2016). Design and generation of Drosophila single guide RNA expression constructs. Cold Spring Harb Protoc 2016(9): pdb prot090779.
  4. Housden, B. E. and Perrimon, N. (2016). Detection of indel mutations in Drosophila by high-resolution melt analysis (HRMA). Cold Spring Harb Protoc 2016(9): pdb prot090795.
  5. Housden, B. E., Valvezan, A. J., Kelley, C., Sopko, R., Hu, Y., Roesel, C., Lin, S., Buckner, M., Tao, R., Yilmazel, B., Mohr, S. E., Manning, B. D. and Perrimon, N. (2015). Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal 8(393): rs9.
  6. Kaelin, W. G. Jr. (2005). The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer 5(9): 689-98.

简介

合成的致死相互作用是一种遗传相互作用,其中两种基因之一的破坏单独具有影响,但它们的组合破坏是致命的。有关合成致死相互作用的知识可以帮助阐明网络结构和确定人类疾病如癌症的候选药物靶标。在果蝇中,组合基因破坏已经通过组合多个RNAi试剂而实现。这里我们通过组合CRISPR和RNAi来描述高通量组合基因破坏的协议。这种方法以前导致了高度可重现和保守的合成致死相互作用的识别(Housden等人,2015)。

背景 遗传相互作用的知识,如合成致死性,对于确定基因之间的功能关系可能是无价的。例如,酵母中的大规模遗传相互作用屏幕最近被用于组装全球“细胞功能的接线图”(Costanzo等人,2016)。或者,可以使用特定类型的遗传相互作用,例如合成致死相互作用来鉴定包括癌症在内的疾病的药物靶标(Kaelin,2005)。
 鉴定合成相互作用需要组合破坏两个基因。以前在果蝇细胞培养中实现该方法的方法是同时递送多个dsRNA试剂(例如,Fisher等人,2015)。然而,RNAi试剂具有局限性,包括脱靶效应和不完全靶标敲低,当多个试剂一起递送时,RNAi试剂复合。通过将CRISPR诱变与单个dsRNA处理相结合,可以避免这些问题,从而更简单地解释筛选结果并强化“点击”识别。

关键字:协同致死, 筛选, RNAi, 果蝇, S2R +细胞, 细胞培养, CRISPR

材料和试剂

  1. 10厘米盘(例如,,Corning,目录号:430167)
  2. T75烧瓶(Corning,目录号:430641U)
  3. 0.2微米过滤器(Thermo Fisher Scientific,目录号:156-4020)
  4. 6孔组织培养板(Corning,目录号:3516)
  5. 96孔透明的底部组织培养板(Corning,目录号:3610)
  6. 40μm细胞过滤器(Corning,Falcon ®,目录号:352340)
  7. 石蜡膜(Parafilm,目录号:PM-999)
  8. 24孔板
  9. 15毫升圆锥管
  10. 提示
  11. S2R +细胞(果蝇基因组资源中心,目录号:150)
  12. sgRNA表达质粒(pl18 - 可根据要求从作者获得)
  13. act-GFP质粒(可根据要求从作者获得)
  14. 化学疗法大肠杆菌细胞(例如,Thermo Fisher Scientific,Invitrogen TM,目录号:C404003)
  15. Effectene Transfection Reagent Kit(QIAGEN,目录号:301427)
  16. 具有1%FBS(GE Healthcare,HyClone TM,目录号:SH30541.03)的PBS(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  17. HRMA试剂(Housden和Perrimon,2016)
  18. 零平坦TOPO PCR克隆试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:450245)
  19. M13F和M13R引物
  20. dsRNA文库在384孔不透明的白色组织培养板中,每孔50ng /μl具有5μldsRNA(参见"果蝇"RNAi筛选中心[ http://fgr.hms.harvard.edu/fly-cell-rnai-libraries ]可用库)。请注意,本协议专注于可从DRSC获得的dsRNA库,但其他库也可从其他来源获取。
  21. CellTiter-Glo试剂(Promega,目录号:G7570)
  22. 施耐德果蝇媒体(Thermo Fisher Scientific,Gibco TM ,目录号:21720)
  23. 青霉素/链霉素(Thermo Fisher Scientific,Invitrogen,目录号:15070063)
  24. 完整的媒体(10%FBS)(见配方)
  25. 高血清培养基(20%FBS)(参见食谱)
  26. 无血清施耐德的媒体(见食谱)

设备

  1. 10毫升移液器
  2. 湿度室(一个装有湿纸巾的tupperware盒子很好)
  3. 25℃的细胞培养箱(任何品牌 - CO 2 规定是不必要的)
  4. 显微镜(例如,,Leica Microsystems,型号:DMIL,具有10倍物镜和任何品牌的相机)
  5. 12通道100微升移液管(例如,Thermo Labsystems)
  6. 12通道10-50微升移液管(例如,,Thermo Labsystems)
  7. 离心机与板式转子(任何品牌)
  8. 巴斯德吸管
  9. 血细胞计数器(任何品牌)
  10. 多渠道水库
  11. 计时器
  12. 旋转器
  13. 具有发光读取能力的读板器(例如,Molecular Devices,型号:SpectraMax范例)
  14. 荧光激活细胞分选仪(例如,BD FACSAria)
  15. 热循环仪(任何品牌)

程序

  1. 制备条件培养基
    1. 在完全培养基中,在10cm皿或T75烧瓶中培养S2R +细胞至100%融合。细胞应在标准条件下培养:25℃,环境CO 2 水平
    2. 使用完全培养基分离细胞1:5,培养3天(或直到几乎100%汇合但不重叠细胞)。
    3. 取出所有介质,并用10 ml的完整介质代替。
    4. 立即通过使用10ml移液管轻轻移液整个培养基来分离细胞,以将细胞从培养皿/烧瓶的底部洗涤,然后将5ml细胞悬浮液转移到新的培养皿或烧瓶中,并加入另外5ml的完全培养基。
    5. 长达16小时(不再)。检查以确保细胞在这一点上看起来健康。
    6. 收集介质并使用0.2μm过滤器进行灭菌以除去所有细胞
    7. 使用高血清培养基稀释培养基1:1。在4°C至4周内储存。

  2. 突变细胞系的产生
    1. 使用如前所述的Pl18载体(Housden等人,2016)设计并产生sgRNA表达质粒。
    2. 将完全培养基中的种子S2R +细胞加入6孔板中,使得细胞在附着(每孔2-8×10 5个细胞/孔在1ml完全培养基中)时为40-80%汇合。允许在25°C附近约20分钟。
    3. 根据标准Effectene转染试剂方案,使用10μlEffectene试剂,从步骤B1转染具有40ng act-GFP质粒和360ng sgRNA表达质粒的细胞。
    4. 在25℃孵育细胞4天
    5. 将100μl条件培养基置于三个透明底部96孔组织培养板的每个孔中
    6. 吸出来自转染细胞的废培养基,并用1ml PBS/1%FBS轻轻移液分离。通过40μm过滤器过滤细胞悬液,以去除细胞聚集体
    7. 使用FACS分离前10%的GFP表达细胞,不包括顶部1%的GFP细胞(通常不可行)。见附注1和图1.
    8. 将每个96孔培养板(将用于在步骤B10中找到焦平面)的100个细胞种子100个孔中,并将单个细胞注入每个平板的剩余的95个孔中。
    9. 用Parafilm密封板边缘,并置于25°C培养箱内的湿度室中。
    10. 孵育板2周,然后在显微镜下检查每个孔以鉴定含有菌落的那些,使用A1将聚焦显微镜。
    11. 对于含有已经生长到足够大小的菌落的孔(参见图2中适合的菌落的例子),通过用100μl移液管轻轻移液,将菌落重新悬浮在已经在孔中的培养基中。将50μl细胞悬浮液与75μl条件培养基混合,并转移到新的96孔板的孔中。使用剩余的50μl细胞悬浮液进行基因组DNA制备分析突变。
    12. 使用先前描述的基因组DNA和HRMA方案鉴定突变菌落(Housden和Perrimon,2016)。
    13. 通过使用M13F和M13R引物从步骤B12测序TOPO克隆的PCR产物来确认突变是纯合的框架。我们建议对每个样品进行至少20个TOPO克隆的构建体测序,以确定靶基因的所有等位基因序列。
    14. 鉴定突变体克隆后,通过从96孔板连续转移到24孔板,然后通过6孔板,最后是10cm培养皿或T75培养瓶将相关细胞群扩大。应使用条件培养基转移细胞,直至达到6孔板,此时可以使用完整培养基。当细胞在每个阶段达到汇合时,应进行转移。


      图1.用于分离单个GFP阳性细胞的FACS设置通过首先选择活细胞(A),然后除去双倍体(B),然后鉴定所有GFP阳性细胞(C),分离GFP阳性细胞,最后选择前10%的GFP表达细胞进行分选(D)。门板层次结构如图E所示

      图2.适用于扩增和序列分析的菌落的实例

  3. dsRNA筛选
    1. 将384孔dsRNA测定板从-80℃储存中除霜并在805x下离心2分钟,然后使用。
    2. 使用巴斯德吸管从T75烧瓶或10厘米培养皿中培养的细胞中除去培养基,同时注意不要分离细胞。请注意,S2R +细胞是粘附的,但在受到干扰时会容易分离
    3. 向每个T75烧瓶或10厘米培养皿中加入10ml无血清培养基至室温。使用此介质通过轻轻移液分离细胞并转移到15 ml锥形管中
    4. 使用血细胞计数器计数细胞,并用新鲜无血清培养基稀释至每毫升500,000个细胞
    5. 从每个测试板上取下密封,将密封件拉开时将板放下。
    6. 将稀释的细胞悬浮液转移到多通道储存器,并将10μl(5,000个细胞)分配到每个384孔dsRNA测定板的每个孔中。每个孔之间不需要改变提示,但应将细胞悬浮液移液到井壁上以避免在井之间转移dsRNA试剂。当第一个单元格添加到第一个板块时启动定时器。
    7. 将离心板短暂(130 x g)持续5秒),将细胞悬浮液移至孔底部,然后置于无菌罩中室温孵育。
    8. 在加入第一组细胞后45分钟,开始向每个孔中加入35μl完全培养基(10%FBS)。以与先前添加的单元格相同的顺序将完整介质添加到板中。
    9. 用Parafilm密封板边缘并放入湿度室。
    10. 在25°C孵育5天。
    11. 向测试板的每个孔中加入30μlCellTiter-Glo试剂,轻轻地将CellTiter-Glo试剂与细胞混合(通过放置在旋转器上或在室温下轻轻摇动5分钟)以使细胞裂解。注意不要过分混合,以免在水中溅起样品或产生气泡。
    12. 使用合适的读卡器读取发光

数据分析

  1. 对于每个板,删除所有边缘阱的数据(见注2),并将记录的发光值除以相应列的中值,然后将新值除以每个相应行的中值。这些变化在移液和正常方差中都会导致人为错误。有关规范化策略的说明,请参见图3

    图3. 36列示例板的列和行的数据归一化。 首先计算每列的中位数(A),然后将每个值除以其列(B)的中位数。然后计算每行转换数据的中值(C),并将每个变换值除以其行(D)的中位数。每个计算的公式都显示在框中的数据点上。

  2. 将板数据布局转换为单列(用于此目的的工具可从 http://www.flyrnai.org/cgi-bin/DRSC_serialize.pl )。将所有值转换为log2以归一化分布。
  3. 计算阴性对照dsRNA试剂(已知非致死基因靶标和非靶向dsRNAs)的平均值和标准偏差(见实例2和图4,对于阴性对照位置的详细信息),每个细胞系中每个重复筛选以鉴别具有高于测定噪声的生存力影响的dsRNA(参见注释3和4)。
  4. 使用数据分析步骤3中针对各个重复和细胞系计算的对照的平均值和标准偏差来计算所有数据点的Z分数:Z =(X-mean)/标准差。请参阅 http://www.statisticshowto.com/probability-and-statistics/z-score/,以便更详细地了解Z分数及其解释
  5. 将命中识别为dsRNA试剂,其在突变细胞系中至少复制的生存力(Z≤-1.5),并且在野生型细胞中的至少⅔重复中没有生存力效应(Z> -1.5)。

笔记

  1. 我们建议您与经验丰富的FACS技术人员进行个别电池的FACS隔离。此外,未经转染的细胞应用于确定自体荧光水平,然后门控分离GFP阳性细胞。
  2. 边缘井是在每个测定板的边缘周围含有水而不是dsRNA试剂的孔。这些包括因为边缘井倾向于具有比中心井更高的变异性。参见果蝇 RNAi筛选中心( http://fgr.hms.harvard.edu/plate-layouts ),了解典型测试板布局的详细信息。图4中以测试板的布局为例。


    图4. 果蝇 RNAi筛选中心测试板的平板布局示例。库之间的布局不同。 GFP和LacZ不影响S2R +细胞的活力,Rho1和Thread是致命的。图片由DRSC提供。

  3. 我们建议您执行三个重复屏幕。然而,考虑到与大规模屏幕相关的高成本,可以进行两次重复以节省时间和资源。在这种情况下,可能需要额外的结果验证才能识别假阳性结果。
  4. 可以仅使用阴性对照样本或整个数据集来计算Z分数。对于小屏幕(小于1,000个基因),我们建议仅使用阴性对照,因为真实命中可能会对整个数据集的标准偏差产生重大影响,从而降低命中呼叫的敏感度。对于大屏幕,例如使用全基因组库,使用整个数据集计算Z分数可以更好地表示数据中的噪声。

食谱

  1. 完成媒体
    施奈德的果蝇媒体
    10%FBS
    1%青霉素/链霉素
  2. 高血清培养基
    施奈德的果蝇媒体
    20%FBS
    1%青霉素/链霉素
  3. 无血清施耐德的媒体
    施奈德的果蝇媒体
    1%青霉素/链霉素

致谢

这里使用的RNAi方案从由果蝇RNAi筛选中心(DRSC)( fgr.hms.harvard.edu )。 Perrimon实验室的工作由NIH和Howard Hughes医学研究所支持。

参考文献

  1. Costanzo,M.,VanderSluis,B.,Koch,EN,Baryshnikova,A.,Pons,C.,Tan,G.,Wang,W.,Usaj,M.,Hanchard,J.,Lee,SD,Pelechano, V.,Styles,EB,Billmann,M.,van Leeuwen,J.,van Dyk,N.,Lin,ZY,Kuzmin,E.,Nelson,J.,Piotrowski,JS,Srikumar,T.,Bahr,S ,Chen,Y.,Deshpande,R.,Kurat,CF,Li,SC,Li,Z.,Usaj,MM,Okada,H.,Pascoe,N.,San Luis,BJ,Sharifpoor,S.,Shuteriqi ,E.,Simpkins,SW,Snider,J.,Suresh,HG,Tan,Y.,Zhu,H.,Malod-Dognin,N.,Janjic,V.,Przulj,N.,Troyanskaya,OG,Stagljar, I.,Xia,T.,Ohya,Y.,Gingras,AC,Raught,B.,Boutros,M.,Steinmetz,LM,Moore,CL,Rosebrock,AP,Caudy,AA,Myers,CL,Andrews,B 。和Boone,C.(2016)。全球遗传相互作用网络映射了细胞功能的接线图。科学 353(6306)。
  2. Fischer,B.,Sandmann,T.,Horn,T.,Billmann,M.,Chaudhary,V.,Huber,W.and Boutros,M。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25748138"target ="_ blank">后生动物细胞中定向遗传相互作用的图。 Elife 4 。
  3. Housden,BE,Hu,Y.和Perrimon,N。(2016)。设计和生成果蝇单指导RNA表达构建体。冷泉哈勃原菌 2016(9):pdb prot090779。
  4. Housden,BE和Perrimon,N。(2016)。通过高分辨率熔解分析(HRMA)检测果蝇中的indel突变。冷春季Harb Protoc 2016(9):pdb prot090795。
  5. Housden,BE,Valvezan,AJ,Kelley,C.,Sopko,R.,Hu,Y.,Roesel,C.,Lin,S.,Buckner,M.,Tao,R.,Yilmazel,B.,Mohr, SE,Manning,BD和Perrimon,N。(2015)。信号 8(393):rs9。
  6. Kaelin,WG Jr.(2005)。< a class ="ke-insertfile"href ="http://xueshu.baidu.com/s?wd=paperuri%3A%28994ef985cfa67e877701ebaa02e16c52%29&filter=sc_long_sign&tn=SE_xueshusource_2kduw22v&sc_vurl=http %3A%2F%2Fpubs.acs.org%2Fservlet%2Flinkout%3Fsuffix%3Dref15%2Fcit15%26dbid%3D8%26doi%3D10.1021%252Fja404868t%26key%3D16110319&ie = utf-8&sc_us = 17908626004135767591"target ="_ blank">在抗癌治疗背景下的合成致死性的概念。 Nat Rev Cancer 5(9):689-98。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Housden, B. E., Nicholson, H. E. and Perrimon, N. (2017). Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells. Bio-protocol 7(3): e2119. DOI: 10.21769/BioProtoc.2119.
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