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Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids
乳腺肿瘤类器官中反义寡核苷酸介导的基因敲低技术

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Abstract

Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids are therefore a promising model system to identify and characterize novel drivers of breast cancer that would be unlikely to be identified using 2D cell lines. Antisense oligonucleotides can be used to efficiently and specifically knockdown target genes in the cell (Bennett et al., 2017). They can be taken up freely by organoids without the need for a transfection agent, making them a convenient tool for routine lab studies and screens.

Keywords: Organoids(类器官), 3D cell culture(3D细胞培养), Mammary tumor(乳腺肿瘤), Breast cancer(乳腺癌), Antisense knockdown(反义敲低), Antisense oligonucleotides(反义寡核苷酸)

Background

Breast cancer is the most frequent malignancy in women worldwide and the second leading cause of cancer mortality in women (Siegel et al., 2017). To improve existing treatment regimens, it is critical to identify and investigate new molecular targets that have the potential to prevent breast cancer progression. We applied RNA-seq to generate a comprehensive catalog of long non-coding RNAs (lncRNAs) that are dysregulated in primary mammary tumors compared to normal mammary epithelial cells and prioritized 30 previously uncharacterized lncRNAs as Mammary Tumor Associated RNAs (MaTARs). In order to functionally assess MaTARs as key drivers of tumor progression, we performed antisense oligonucleotide (ASO)-mediated knockdown assays of all 30 MaTARs in 3D mammary tumor organoids (Diermeier et al., 2016).
   ASOs are short (20-mers), single stranded DNA molecules containing phosphorothioate-modified nucleotides as well as modifications of the 2’-ribose (5-10-5 2’-MOE gapmer) (Geary et al., 2015). Upon binding of the ASO to its complementary target, the RNA-DNA duplex stimulates degradation of the lncRNA by RNase H and thereby reduces the level of the respective transcript (Wu et al., 2004). Importantly, we found that ASO uptake in primary mammary tumor cells and organoids is efficient without the use of transfection agents, a mechanism that has been studied in detail in hepatocytes (Koller et al., 2011). ASO-mediated knockdown is particularly efficient for nuclear retained lncRNAs (Lennox and Behlke, 2016).
   Organoids represent an ex vivo model of mammary gland development and model branching morphogenesis in 3D culture (Ewald, 2013; Fata et al., 2007), which is driven by two physiological processes: collective cell migration and cell proliferation. As the same processes also drive tumor invasion, the mammary organoid system can be utilized to model invasive breast cancer in vitro. Loss of branching was observed upon ASO-mediated knockdown of 20 MaTARs in organoids (Diermeier et al., 2016) as well as the lncRNA Malat1 (Arun et al., 2016), indicating that these RNAs are involved in mammary tumor cell proliferation and/or collective cell migration. Hence, we suggest that antisense-mediated knockdown in mammary tumor organoids can be used to identify and characterize novel drivers of tumor progression.

Materials and Reagents

  1. 5 cm sterile cell culture dish (e.g., Corning, Falcon®, catalog number: 353002 )
  2. Optional: Disposable plastic Cryomold (e.g., Tissue-Tek Cryomold, Electron Microscopy Sciences, catalog number: 62534-25 )
  3. 15 ml and 50 ml centrifuge tubes (e.g., Crystalgen, catalog number: 23-2265 ; Corning, Falcon®, catalog number: 352098 )
  4. 24-well cell culture plate (Greiner Bio One International, catalog number: 662160 )
  5. Cell culture flask, 75 cm2 (e.g., Corning, Falcon®, catalog number: 353136 )
  6. Pasteur glass pipettes (e.g., Kimble Chase Life Science and Research Products, catalog number: 63A54 )
  7. Sterile pipette tips (e.g., Corning)
  8. Cell strainer 70 micron (e.g., Corning, catalog number: 431751 )
  9. 0.2 ml PCR strip tubes (e.g., Corning, Axygen®, catalog number: PCR-0208-CP-C )
  10. 96-well reaction plates (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4346907 )
  11. Optical adhesive film (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4311971 )
  12. Mammary tumor-bearing mouse (e.g., MMTV-PyMT (Guy et al., 1992)) with palpable tumors. Optimal tumor size is ~5-10 mm in diameter
  13. 200 Proof ethyl alcohol (e.g., UltraPure, catalog number: 200CSPTP )
  14. Sterile water
  15. Ice
  16. Matrigel Growth Factor reduced Basement Membrane Matrix, phenol-red free (Corning, catalog number: 356231 )
  17. Optional: Tissue-Tek O.C.T. compound (Electron Microscopy Sciences, catalog number: 62550-12 )
  18. Liquid N2
  19. Cell recovery solution (Corning, catalog number: 354253 )
  20. 1x DPBS (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  21. TRIzol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596018 )
  22. GlycoBlue (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9516 )
  23. DNAse I, amplification grade, for cDNA synthesis (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: 18068015 )
  24. Ethylenediaminetetraacetate acid disodium salt (EDTA)
  25. Fetal bovine serum (FBS) (e.g., VWR, product number: 1500-500 )
  26. Gentamicin 50 mg/ml (e.g., Lonza, catalog number: 17-528Z )
  27. Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I1882-100MG )
  28. Trypsin (e.g., Mediatech, catalog number: 25-054-CI )
  29. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138-1G )
  30. Advanced DMEM/F12 (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 12634010 )
  31. Bovine serum albumin (BSA) (e.g., Sigma-Aldrich, catalog number: A2153-100G )
  32. DNase I from bovine pancreas for organoid preparation (e.g., Sigma-Aldrich, catalog number: D4263-1VL )
  33. Pen/Strep (e.g., Sigma-Aldrich, catalog numbers: PENNA-100MU and S6501-100G )
  34. ITS liquid media supplement 100x (Sigma-Aldrich, catalog number: I3146-5ML )
  35. Murine FGF-basic (PeproTech, catalog number: 450-33 )
  36. Dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, catalog number: D2650-5x10ML )
  37. Nuclease-free water (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
  38. TaqMan Reverse Transcription Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: N8080234 )
  39. PowerUp SYBR Green Master Mix (Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: A25743 )
  40. Chloroform, purified (e.g., Avantor Performance Materials, MACRON, catalog number: 4432-10 )
  41. Isopropanol, molecular biology grade (e.g., Fisher Scientific, catalog number: BP2618500 )
  42. Collagenase solution (see Recipes)
  43. BSA solution (see Recipes)
  44. DNase solution (see Recipes)
  45. Organoid medium (10 ml, sufficient for 10 wells) (see Recipes)
  46. Freezing medium (see Recipes)
  47. cDNA Master mix (see Recipes)
  48. qPCR Master mix (see Recipes)

Equipment

  1. Biological safety cabinet (e.g., NuAire)
  2. Dissection tools (e.g., 114.3 mm scissors, Sklar Surgical Instruments, catalog number: 98-104 ; forceps, Sklar Surgical Instruments, catalog number: 97-751 , sterile scalpels, e.g., Sklar Surgical Instruments, catalog number: 06-3110 )
  3. Cell culture incubator (e.g., Heracell i Copper CO2 incubator, Thermo Fischer Scientific, Thermo ScientificTM, model: HeracellTM 150i and 240i , catalog number: 50116050)
  4. Shaker with temperature control (e.g., Thomas Scientific, catalog number: 1222U12)
    Manufacturer: Benchmark Scientific, catalog number: H1000-M .
  5. Centrifuge for 15 and 50 ml tubes (e.g., Eppendorf, model: 5804 )
  6. Centrifuge for 1.5 ml reaction tubes with cooling function (e.g., Eppendorf, model: 5427 R )
  7. Vacuum suction
  8. Micro-pipettes (e.g., Gilson, catalog number: F167300 , catalog number for Thomas Scientific: 1222N73)
  9. Phase-contrast microscope (e.g., Nikon)
  10. Optional: Cryo-Safe Freeze Controller (e.g., SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F18844-0000 )
  11. NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)
  12. PCR machine (e.g., Applied Biosystems Proflex Thermocycler, Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4484073 )
  13. qPCR machine (e.g., Applied Biosystems StepOne Plus Real-Time PCR system, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4376600 )
  14. -80 °C freezer (e.g., VWR, catalog number: 10160-728 )
  15. Water bath (e.g., PolyScience, catalog number: WB10A11B )
  16. Optional: liquid N2 freezers (e.g., VWR, catalog number: 82017-934 )
  17. Refrigerator (4 °C)

Procedure

  1. Design of antisense oligonucleotides (ASOs)
    1. Choose a target gene for knockdown. The protocol described here is specifically suitable for long non-coding RNAs, but knockdown of protein-coding transcripts is equally possible.
    2. Design 20-mers complimentary to the RNA sequence. Usually, ASOs are designed complimentary to exons, but in some cases ASOs targeting introns can be efficient as well. Design at least two specific ASOs (100% sequence identity) for the target sequence as well as a negative control (e.g., a scrambled ASO control, see (Diermeier et al., 2016)). It is recommended to start with up to ten individual ASOs for new targets, as not every ASO will result in an efficient knockdown.
    3. Use Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure the ASOs are specific to the intended target only. Stringency is important at this step–even one or two mismatches can result in unwanted off-target effects.
    4. ASOs used in knockdown experiments carry modified nucleotides to increase binding affinity. Of the 20 nucleotides, the first and last 5 nucleotides are modified with 2’-O-methyoxy-ethyl (MOE). The 10 nucleotides in the middle remain unmodified. In the US, MOE modifications can be ordered e.g., from Integrated DNA Technologies (IDT).
    5. Upon arrival, resuspend lyophilized ASOs in sterile DPBS. Working stock concentration: 200 µM.
    6. Store at 4 °C for short-term storage (up to one week) and -20 °C for long-term storage. Aliquot to avoid repeated freeze-thaw cycles.

  2. Preparation of mouse mammary tumor organoids
    1. Euthanize mammary tumor-bearing mouse according to the local regulations.
    2. Pin mouse with abdominal side up to dissection board, spray down mouse with 70% ethanol and transfer to sterile cell culture hood. All steps from here on are to be performed under sterile conditions.
    3. Remove tumor using sterile dissection tools and place it in a 5 cm cell culture dish. In case of multiple tumors, prepare organoids from each tumor separately.
    4. Remove necrotic tissue from tumor if present using a scalpel. In general, necrotic tissue is darker and softer compared to the surrounding solid tumor. Necrotic tissue can often be found as dark liquefied tissue at the center of large tumors (Morton and Houghton, 2007).
    5. Optional: It is recommended to store a small part of the tumor for histological analysis. Therefore, use a scalpel and carefully cut about ¼ of the tumor. Embed the tumor in O.C.T. in a plastic Cryomold and store at -80 °C.
    6. Cut the remaining tumor tissue with a scalpel until the tissue pieces are about 2-3 mm in diameter.
    7. Transfer the tumor tissue to a 50 ml tube containing 25 ml of collagenase solution (Recipe 1).
    8. Shake the collagenase solution containing tumor tissue in an incubator at 100 rpm at 37 °C for 45 min. The suspension now appears cloudy. Over-digestion leads to a large amount of single cells, which should be avoided.
    9. Pre-warm DMEM/F12 medium and organoid medium to RT.
    10. Pre-warm a 24-well cell culture plate (see Notes 1 and 2) and a 75 cm2 cell culture flask filled with sterile water to 37 °C.
    11. Thaw Matrigel on ice.
    12. Centrifuge tube with collagenase solution at 520 x g for 10 min at 25 °C.
    13. Aspirate the supernatant using a Pasteur glass pipette connected to vacuum suction, leaving about 1 ml of medium on top of the pellet.
    14. Pre-coat a 10 ml sterile pipette tip by pipetting BSA solution (Recipe 2) up and down once.
      Important: All plastic pipettes and tubes should be pre-coated with BSA solution immediately before use from this point on to avoid sticking of organoids to the plastic surface, unless otherwise specified.
    15. Resuspend the pellet in 10 ml of pre-warmed DMEM/F12 medium using the pre-coated pipette tip.
    16. Transfer the suspension to a 15 ml centrifugation tube that has been pre-coated with BSA solution.
    17. Centrifuge tube at 520 x g for 10 min at 25 °C.
    18. Aspirate the supernatant as described in step B13. Pasteur pipettes need not be pre-coated with BSA before use.
    19. Resuspend the pellet in 4 ml of DNase solution (Recipe 3) using a pre-coated 10 ml pipette tip.
    20. Gently rock by hand for 2-5 min.
    21. Add 6 ml of DMEM/F12 and resuspend thoroughly with a pre-coated 10 ml pipette tip.
    22. Filter the suspension through a cell strainer into a fresh, pre-coated 15 ml tube to remove large chunks that remain after collagenase digestion.
    23. Centrifuge tube at 520 x g for 10 min at 25 °C.
    24. Aspirate the supernatant as described in step B13. Pasteur pipettes need not be pre-coated with BSA before use.
    25. Resuspend the pellet in 10 ml of DMEM/F12 medium using a pre-coated pipette tip.
    26. Pulse-centrifuge tube to 520 x g at 25 °C. Stop the centrifuge as soon as it reaches 520 x g.
    27. Repeat steps B25-B27 three more times (for a total of four times) or until the medium after puls-centrifugation is clear. At the last aspiration step, carefully remove the entire medium.
    28. Resuspend the pellet in 10 ml of DMEM/F12 medium using a pre-coated pipette tip.
    29. Make sure the suspension is well mixed. Remove 50 µl using a micropipette and transfer to a sterile 5 cm cell culture dish to determine the density of organoids in solution.
    30. Count the number of organoids in the 50 µl drop under a microscope. Organoids are counted in the dish, no cover slips are required. An example of a mammary tumor organoid right after preparation is shown in Figure 1. Depending on the size of the tumor, the total amount of organoids per preparation can vary from approximately 5,000 to 40,000. Organoids that are not needed for the current experiment can be frozen and stored as described in step B42.


      Figure 1. Branching morphogenesis of organoids. In this example, organoids were prepared from MMTV-PyMT derived mammary tumors. Left panel: Organoids at day 0, right after organoid preparation, embedded in Matrigel. Right panel: Organoids at day 6, grown in the presence of FGF-basic, embedded in Matrigel.

    31. Centrifuge tube at 520 x g for 2 min at 25 °C.
    32. Aspirate the supernatant completely and carefully using a Pasteur glass pipette. Pasteur pipettes need not be pre-coated with BSA before use.
    33. Resuspend the organoid pellet in Matrigel based on the following criteria and put on ice immediately. Pipette tips and cell culture plates need not be pre-coated with BSA for this step due to the high protein content of Matrigel. To plate organoids in 24-well cell culture plates, 80 µl are required per well. Organoid plating density varies depending on the downstream assay. For microscopy analysis, plate organoids at a density of 2 organoids/µl (see Note 2). For RNA extractions, plate organoids at a density of 5 organoids/µl. One well yields a sufficient amount of RNA to perform qRT-PCR assays downstream; if higher RNA yields are required, pool two or more wells when extracting RNA (see Procedure C: RNA extraction from organoids).
    34. Prepare a sterile, pre-warmed (37 °C) 24-well cell culture plate and place it on top of a pre-warmed (37 °C) 75 cm2 cell culture flask filled with sterile water. The water-filled flask acts like a heated plate and is crucial for quick solidification of Matrigel.
    35. Carefully plate the Matrigel containing organoids as 80 µl domes in the middle of the wells (see Note 1). Avoid air bubbles. Pipette tips need not be pre-coated with BSA for this step.
    36. Incubate the plates in a cell culture incubator at 5% CO2 and 37 °C for 20 min.
    37. Add 1 ml of pre-warmed (RT) organoid medium (Recipe 4) per well. Be careful not to disturb the Matrigel domes.
    38. Add antisense oligonucleotides (ASOs) to the medium under sterile conditions. ASOs are added individually, one per well. Optimal ASO concentrations vary depending on the ASO and the target gene. A titration in the range from 0.1-5 µM should be performed (see Note 3). As a starting point, a concentration of 5 µM per well (25 µl of a 200 µM ASO solution) should result in a > 50% knockdown for most targets. ASOs are taken up freely by mammary organoids, no transfection reagent is required.
    39. Leave at least one well untreated and treat at least one well with a scrambled ASO (scASO) as negative controls.
    40. Incubate organoids in a cell culture incubator at 5% CO2 and 37 °C.
    41. Change the medium to fresh organoid medium and replenish ASOs three days past organoid generation. Organoids should be harvested no later than 6 days post generation (see Note 4). An example of a mammary organoid cultured for 6 days in the presence of FGF is shown in Figure 1.
    42. Optional: Any extra organoids that are not needed for the current experiment can be frozen and stored. To do so, calculate the amount of required organoids and respective volume of the organoid suspension at step B30. Split the organoid suspension in two aliquots, one containing the amount needed for the experiment and the other containing any additional organoids. Proceed with both aliquots as described in steps B31 and B32. Resuspend the organoids needed for the experiment in Matrigel and proceed as described above. Resuspend the extra organoids in freezing medium (Recipe 5), aliquot in cryovials and transfer to a CryoSafe freeze controller. Store at -80 °C overnight and transfer to liquid N2 the next day. To thaw organoids, warm the vial to 37 °C and resuspend the organoids in 10 ml of DMEM. Follow steps B31 to B37 to plate the organoids in Matrigel. For thawed organoids, a seeding density of 5-10 organoids/µl is recommended, as not all organoids will survive the freeze/thaw process.

  3. RNA extraction from organoids
    1. Organoids can be harvested as early as 24 h post ASO addition to assay the knockdown efficiency. If phenotypic analysis is required as well, live-cell imaging can be performed from day 3-day 6 after ASO addition (the day of organoid preparation is day 0), as the branching morphogenesis starts around day 3-4. Alternatively, endpoint microscopic analysis can be performed on day 6 to compare ASO treated organoids to control organoids. In this case, knockdown efficiency can still be assayed on day 6 (see Note 4).
    2. Transfer the 24-well plate containing organoids from the cell culture incubator to the refrigerator (4 °C). This step will cause the Matrigel to liquefy over night.
    3. The next day, transfer the content of each well (medium and Matrigel) to a 15 ml centrifugation tube on ice.
    4. Add 200 µl of cold cell recovery solution to each well to depolymerize any residual Matrigel and incubate for 1 h at 4 °C.
    5. Transfer the content of each well (cell recovery solution and Matrigel) and combine with the previously removed organoid/Matrigel mix. If Matrigel sticks to the bottom, careful scraping may be used to detach Matrigel and organoids.
    6. Incubate on ice for 1 h.
    7. Centrifuge at 420 x g for 10 min at 4 °C.
    8. Aspirate the supernatant carefully, using a micropipette.
    9. Resuspend the organoid pellet in 100 µl of cold cell recovery solution.
    10. Incubate on ice for 10 min.
    11. Centrifuge at 420 x g for 5 min at 4 °C.
    12. Aspirate the supernatant carefully, using a micropipette.
    13. Resuspend the pellet in 1 ml of 1x DPBS (RNase-free).
    14. Centrifuge at 420 x g for 5 min at 4 °C.
    15. Aspirate the supernatant carefully, using a micropipette.
    16. Resuspend the cell pellet in 1 ml TRIzol.
    17. Isolate RNA from organoids according to the manufacturer’s instructions. Addition of glycogen (such as GlycoBlue) is recommended, as the RNA yield can be low if organoids were seeded at low density and harvested after 24 h. Resuspend RNA pellet in 10-20 µl of nuclease-free water.

  4. cDNA synthesis and qPCR to analyze knockdown efficiency
    1. Measure the RNA concentration using a NanoDrop 2000 UV-Vis spectrophotometer. Keep RNA on ice at all times to avoid degradation.
    2. Use a total of 1,000 ng RNA in a volume of 8 µl (concentrations of 125 ng RNA/µl). If concentrations are higher, dilute in nuclease-free water.
    3. Transfer sample to 0.2 ml PCR tubes.
    4. Perform DNase digestion to remove any potentially co-purified genomic DNA by adding 1 µl of 10x DNase reaction buffer and 1 µl DNase I to the RNA sample (total volume: 10 µl).
    5. Mix reaction by gently pipetting up and down. Spin briefly in a centrifuge.
    6. Incubate reaction at 25 °C in a PCR machine for 15 min.
    7. Add 1 µl of 25 mM EDTA.
    8. Mix reaction by gently pipetting up and down. Spin briefly in a centrifuge.
    9. Inactivate reaction at 65 °C in a PCR cycler for 10 min.
    10. Transfer samples on ice immediately after the 10 min incubation is completed. Do not leave samples in PCR machine while ramping down to RT.
    11. Prepare cDNA Master mix (Recipe 6).
    12. Add 39 µl of cDNA Master mix to samples on ice.
    13. Mix reaction by gently pipetting up and down. Spin briefly in a centrifuge.
    14. Perform the following incubation in a PCR machine:
      25 °C for 10 min
      48 °C for 30 min
      95 °C for 5 min
    15. cDNA can be stored short-term at 4 °C or long-term at -20 °C.
    16. Prepare qPCR Master mix (Recipe 7) containing primers for the target gene (see Note 5) as well as qPCR Master mix containing primers for an internal control (‘housekeeping’) gene. When calculating the amount of Master mix needed, consider that samples will be pipetted in triplicates.
    17. Commonly used genes for internal controls include beta-actin, GAPDH or ribosomal protein genes. Internal controls should be chosen carefully; their expression level should be consistent and independent on experimental conditions such as ASO treatments. The expression level of an internal control gene should be in the same range as the target gene. General rules regarding qPCR experiments and qPCR primer design can be found in (Bustin et al., 2009).
    18. Prepare a 96-well plate on ice.
    19. Pipette 2 µl of cDNA into one well of the 96-well plate. Each sample will be pipetted in triplicates for both the target and the housekeeping gene (= 6 wells containing 2 µl of cDNA per sample).
    20. Add 18 µl of Master mix containing either target gene or internal control gene primers to the wells.
    21. Seal the 96-well plate with an optical adhesive film. Avoid touching the film with your fingers, handle carefully by the edges.
    22. Spin down the plate at 100 x g for 1 min at RT.
    23. Place the plate in the qPCR machine and run the following program:
      95 °C for 10 min
      40 cycles of: 95 °C for 15 sec
      60 °C for 60 sec
      Followed by
      Melt curve 95 °C → 60 °C, 1 °C/min

Data analysis

  1. Export Ct values from the qPCR machine and analyze using the 2-ΔΔCt Method (Livak and Schmittgen, 2001). There should be a noticeable reduction of the target gene in samples treated with ASOs compared to untreated samples and samples treated with scASO as negative control. The scASO should not have any effect on gene expression and result in values very similar to untreated samples. Very potent ASOs can result in a > 90% knockdown efficiency, but knockdown efficiencies vary between different ASOs and target genes. At least three independent biological replicate should be performed for statistically meaningful results. Insufficient knockdown efficiencies can be improved by increasing the amount of ASOs used, testing ASOs targeting different regions of the RNA and varying organoid densities. In addition, ASOs can be mixed with Matrigel when plating the organoids and also added to the medium for maximum knockdown efficiency.
  2. Endpoint analysis of phenotypic changes in organoids can be performed using standard light microcopy. When comparing ASO-treated organoids to control organoids, changes in the number, size and branching morphogenesis are often observed. To perform statistically significant comparisons, at least 100 organoids per treatment should be counted per replicate, with a minimum of three experimental replicates.

Notes

  1. Organoids can also be grown in 48-well and 96-well plates for assays requiring higher throughput. The size for the Matrigel domes has to be scaled down accordingly, e.g., 40 µl/well in 48-well plates and 20 µl/well in 96-well plates.
  2. If live-cell imaging is to be performed to analyze organoids, use glass-bottom plates (e.g., MatTek)
  3. If using new, untested ASOs, it is recommended to test several concentrations (e.g., 0.1, 0.5, 1 and 5 µM) and compare the knockdown efficiency.
  4. According to our RNA-seq data, culturing of organoids in an artificial extracellular matrix (ECM) for up to seven days does not significantly alter the expression patterns of lncRNAs (Diermeier et al., 2016).
  5. For optimal results, primers for qPCR should not overlap with the ASO binding site, and the ASO should not bind within the qPCR amplified region.

Recipes

  1. Collagenase solution (50 ml, sufficient for two tumors)
    2 ml FBS
    2.5 µl gentamycin (50 mg/ml)
    25 µl insulin
    4 ml trypsin
    75 mg collagenase
    Add DMEM/F12 up to 50 ml
    Sterile filter before use
    Use immediately after preparation
  2. BSA solution (2.5%)
    Dissolve 2.5 mg BSA in 100 ml DPBS
    Sterile filter before use
    Can be re-used and stored at 4 °C for up to four weeks
  3. DNase solution
    Add 40 µl of DNase I from bovine pancreas (1 U/µl) to 10 ml of DMEM/F12 medium
    Use immediately after preparation
  4. Organoid medium (10 ml, sufficient for 10 wells)
    100 µl Pen/strep
    100 µl ITS
    3 µl FGF-basic
    Add DMEM/F12 up to 10 ml
    Can be stored at 4 °C for one week
    Without addition of FGF2, organoid medium can be stored at 4 °C for up to four weeks
  5. Freezing medium
    10% DMSO in FBS
    Can be stored long-term at -20 °C
  6. cDNA Master mix (all components except for nuclease-free H2O are included in the TaqMan Reverse Transcription Kit)
    10x TaqMan RT buffer
    5 µl
    Random hexamers
    2.5 µl
    MgCl2 (25 mM)
    11 µl
    dNTPs
    10 µl
    RNase Inhibitor  
    1 µl
    RTase 
    1.25 µl
    Nuclease-free H2
    8.25 µl
    Total volume  
    39 µl
    Use immediately after preparation
  7. qPCR Master mix
    PowerUp SYBR Green Master mix
    10 µl
    Nuclease-free H2O              
    7 µl
    Total 
    17 µl
    Primer mix (for + rev, 10 µM)
    1/well (conc.: 0.5 µM/well)
    Use immediately after preparation

Acknowledgments

Our tumor organoid protocol is based on previous work from Andrew Ewald’s lab (Ewald, 2013; Nguyen-Ngoc et al., 2015). The Manhasset Women’s Coalition Against Breast Cancer (S.D.D.) and the NCI 5P01CA013106-Project 3 (D.L.S.) supported this research.

References

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简介

在3D生长的原发性乳腺肿瘤组织是研究肿瘤生物学的优秀系统。 它们类似于天然上皮的组织和生理学,比2D生长的癌细胞系更为紧密,另外还与ECM的模型相互作用(Boj et al。,2015; Clevers,2016; Shamir and Ewald,2014)。 因此,乳腺肿瘤组织因子是一种有希望的模型系统,用于识别和表征不可能使用2D细胞系识别的乳腺癌的新型驱动因素。 反义寡核苷酸可用于有效和特异地敲低细胞中的靶基因(Bennett等,2017)。 它们可以被有机物自由摄取,而不需要转染剂,使其成为常规实验室研究和筛选的便捷工具。
【背景】乳腺癌是全世界妇女中最常见的恶性肿瘤,是妇女癌症死亡率的第二大原因(Siegel等,2017)。为了改善现有的治疗方案,确定和调查具有预防乳腺癌进展潜力的新分子靶标至关重要。我们应用RNA-seq来产生与正常乳腺上皮细胞相比在原发性乳腺肿瘤中失调的长非编码RNA(lncRNA)的综合目录,并将30个先前未表征的lncRNA作为乳腺肿瘤相关RNA(MaTARs)进行优先排序。为了功能评估MaTARs作为肿瘤进展的关键驱动因素,我们对3D乳腺肿瘤组织中的所有30个MaTARs进行了反义寡核苷酸(ASO)介导的敲低分析(Diermeier等,2016)。
   ASO是短(20-mers),含有硫代磷酸酯修饰的核苷酸的单链DNA分子以及2'-ribose(5-10-5 2'-MOE gapmer)的修饰(Geary等,2015)。在将ASO与其互补靶标结合后,RNA-DNA双链体通过RNA酶H刺激lncRNA的降解,从而降低各个转录物的水平(Wu等,2004)。重要的是,我们发现,原代乳腺肿瘤细胞和组织中的ASO摄取是有效的,而不需要使用转染试剂,这种机制已经在肝细胞中进行了详细的研究(Koller等,2011)。 ASO介导的敲低对于核保留的lncRNA是特别有效的(Lennox和Behlke,2016)。
   有机体代表了三维培养中乳腺发育和模型分支形态发生的离体模型(Ewald,2013; Fata et al。,2007),由两个生理过程驱动:集体细胞迁移和细胞增殖。由于相同的过程也可以驱使肿瘤侵袭,乳腺组织系统可用于体外侵袭性乳腺癌模型。在ASO介导的组织中的20MTAR(Diermeier等,2016)的敲除以及lncRNA Malat1(Arun等,2016)中观察到分支损失,表明这些RNA涉及乳腺肿瘤细胞增殖, /或集体细胞迁移。因此,我们建议乳腺肿瘤组织中的反义介导的敲低可用于鉴定和表征肿瘤进展的新型驱动因素。

关键字:类器官, 3D细胞培养, 乳腺肿瘤, 乳腺癌, 反义敲低, 反义寡核苷酸

材料和试剂

  1. 5cm无菌细胞培养皿(例如,Corning,Falcon ,目录号:353002)
  2. 可选的:一次性塑料冷冻模型(例如,Tissue-Tek Cryomold,Electron Microscopy Sciences,目录号:62534-25)
  3. 15ml和50ml离心管(例如,Crystalgen,目录号:23-2265; Corning,Falcon,目录号:352098)
  4. 24孔细胞培养板(Greiner Bio One International,目录号:662160)
  5. 细胞培养瓶,75cm 2(例如,Corning,Falcon,目录号:353136)
  6. 巴斯德玻璃移液器(例如,Kimble Chase Life Science and Research Products,目录号:63A54)
  7. 无菌移液器吸头(例如,,康宁)
  8. 细胞过滤器70微米(例如,Corning,目录号:431751)
  9. 0.2ml PCR条带管(例如,Corning,Axygen,目录号:PCR-0208-CP-C)
  10. 96孔反应板(例如,Thermo Fischer Scientific,Applied Biosystems TM,目录号:4346907)
  11. 光学粘合膜(例如,Thermo Fischer Scientific,Applied Biosystems TM,目录号:4311971)
  12. 具有可触知肿瘤的乳腺肿瘤小鼠(例如,MMTV-PyMT(Guy等人,1992)))。最佳肿瘤大小直径约5-10毫米
  13. 200证明乙醇(例如UltraPure,目录号:200CSPTP)
  14. 无菌水

  15. Matrigel生长因子降低基底膜基质,无酚红(Corning,目录号:356231)
  16. 可选:Tissue-Tek O.C.T.化合物(Electron Microscopy Sciences,目录号:62550-12)
  17. 液体N 2
  18. 细胞回收溶液(Corning,目录号:354253)
  19. 1x DPBS(例如,Thermo Fisher Scientific,Gibco TM,目录号:14190250)
  20. TRIzol(Thermo Fisher Scientific,Invitrogen TM,目录号:15596018)
  21. GlycoBlue(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9516)
  22. 用于cDNA合成的DNA酶I,扩增级,例如Thermo Fisher Scientific,Invitrogen,目录号:18068015)
  23. 乙二胺四乙酸二钠盐(EDTA)
  24. 胎牛血清(FBS)(例如,VWR,产品编号:1500-500)
  25. 庆大霉素50mg / ml(例如,Lonza,目录号:17-528Z)
  26. 来自牛胰脏的胰岛素(Sigma-Aldrich,目录号:I1882-100MG)
  27. 胰蛋白酶(例如, Mediatech,目录号:25-054-CI)
  28. 来自
  29. 高级DMEM / F12(例如,Thermo Fisher Scientific,Gibco TM,目录号:12634010)
  30. 牛血清白蛋白(BSA)(例如,Sigma-Aldrich,目录号:A2153-100G)
  31. 来自牛胰腺的DNase I用于组织制剂(例如,Sigma-Aldrich,目录号:D4263-1VL)
  32. Pen / Strep(例如,,Sigma-Aldrich,目录号:PENNA-100MU和S6501-100G)
  33. ITS液体培养基补充剂100x(Sigma-Aldrich,目录号:I3146-5ML)
  34. Murine FGF-basic(PeproTech,目录号:450-33)
  35. 二甲基亚砜(DMSO)(例如,Sigma-Aldrich,目录号:D2650-5x10ML)
  36. 不含核酸酶的水(例如,Thermo Fisher Scientific,Invitrogen,Supest TM,目录号:AM9937)
  37. TaqMan逆转录试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:N8080234)
  38. PowerUp SYBR Green Master Mix(Thermo Fischer Scientific,Applied Biosystems TM ,目录号:A25743)
  39. 氯仿,纯化(例如,Avantor Performance Materials,MACRON,目录号:4432-10)
  40. 异丙醇,分子生物学级(例如,Fisher Scientific,目录号:BP2618500)
  41. 胶原酶溶液(参见食谱)
  42. BSA溶液(参见食谱)
  43. DNase解决方案(见配方)
  44. 有机体培养基(10 ml,足够10孔)(参见食谱)
  45. 冷冻介质(参见食谱)
  46. cDNA主混合物(参见食谱)
  47. qPCR主混合(见配方)

设备

  1. 生物安全柜(例如,,NuAire)
  2. 解剖工具(例如,114.3mm剪刀,Sklar手术器械,目录号:98-104;镊子,Sklar手术器械,目录号:97-751,无菌解剖刀,例如,Sklar Surgical Instruments,目录号:06-3110)
  3. 细胞培养培养箱(例如,,Heracell i Copper CO 培养箱,Thermo Fischer Scientific,Thermo Scientific TM,型号:Heracell 150i和240i,目录号:50116050)
  4. 具有温度控制的振动器(例如,,Thomas Scientific,目录号:1222U12)
    制造商:Benchmark Scientific,目录号:H1000-M。
  5. 离心机用于15和50ml管(例如,Eppendorf,型号:5804)
  6. 离心机用于具有冷却功能的1.5ml反应管(例如,Eppendorf,型号:5427R)
  7. 真空吸力
  8. 微量移液管(例如,Gilson,目录号:F167300,Thomas Scientific的目录号:1222N73)
  9. 相位显微镜(例如,尼康)
  10. 可选:Cryo-Safe Freeze控制器(例如,,SP Scienceware - Bel-Art产品 - H-B仪器,目录号:F18844-0000)
  11. NanoDrop 2000紫外 - 可见分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM,目录号:ND-2000)
  12. PCR机器(例如,Applied Biosystems Proflex Thermocycler,Thermo Fisher Scientific,Applied Biosystems TM,目录号:4484073)
  13. qPCR机器(例如,Applied Biosystems StepOne Plus Real-Time PCR系统,Thermo Fisher Scientific,Thermo Scientific TM,目录号:4376600)
  14. -80℃冷冻机(例如,,VWR,目录号:10160-728)
  15. 水浴(例如,,PolyScience,目录号:WB10A11B)
  16. 可选:液体N 2冷冻箱(例如,VWR,目录号:82017-934)
  17. 冰箱(4°C)

程序

  1. 反义寡核苷酸(ASO)的设计
    1. 选择一个靶基因进行击倒。这里描述的方案特别适用于长非编码RNA,但蛋白编码转录物的敲除同样是可能的。
    2. 设计与RNA序列相互补充的20-mers。通常,ASO被设计为与外显子互补,但在某些情况下,针对内含子的ASO也是有效的。为目标序列设计至少两个特定的ASO(100%序列同一性)以及一个负调控(例如,一个加扰的ASO控制),参见(Diermeier等人 ,2016))。建议最多使用十个独立的ASO进行新的目标,因为不是每个ASO都会导致有效的击倒。
    3. 使用核苷酸BLAST( https://blast.ncbi.nlm。 nih.gov/Blast.cgi ),以确保ASO仅针对目标目标。严格性在这一步很重要 - 即使一个或两个不匹配可能会导致不想要的脱靶效果。
    4. 用于敲除实验的ASOs携带经修饰的核苷酸以增加结合亲和力。在20个核苷酸中,用2'-O-甲氧基 - 乙基(MOE)修饰第一个和最后5个核苷酸。中间的10个核苷酸保持未修饰。在美国,可从Integrated DNA Technologies(IDT)订购MOE修改例如。
    5. 抵达后,将无菌DPBS中的冻干ASO重新悬浮。工作库存浓度:200μM
    6. 储存于4°C,短期储存(长达一周)和-20°C长期储存。等分试样以避免反复冻融循环。

  2. 小鼠乳腺肿瘤组织的制备
    1. 按照当地法规安乐死乳腺肿瘤小鼠。
    2. 将小鼠腹部侧向上切割板,用70%乙醇喷雾小鼠,转移到无菌细胞培养罩。从这里开始的所有步骤都将在无菌条件下进行。
    3. 使用无菌解剖工具去除肿瘤,并将其置于5cm细胞培养皿中。在多发肿瘤的情况下,分别从每个肿瘤中制备组织细胞
    4. 使用手术刀从肿瘤中去除坏死组织。一般来说,与周围的实体瘤相比,坏死组织更暗和更柔软。坏死组织通常可以作为大肿瘤中心的黑色液化组织发现(Morton和Houghton,2007)。
    5. 可选:建议存储少部分肿瘤用于组织学分析。因此,使用手术刀仔细切开肿瘤的1/4。将肿瘤嵌入O.C.T.在塑料冷藏库中,储存在-80°C。
    6. 用手术刀切割剩余的肿瘤组织,直到组织块直径约2-3毫米
    7. 将肿瘤组织转移到含有25ml胶原酶溶液(配方1)的50ml管中
    8. 在培养箱中以100rpm的速度在37℃下摇动包含肿瘤组织的胶原酶溶液37℃45分钟。暂停现在显得多云。过度消化会导致大量单细胞,应避免使用。
    9. 预热DMEM / F12培养基和有机体培养基至RT。
    10. 预先将装有无菌水的24孔细胞培养板(参见注释1和2)和75厘米 2细胞培养瓶预温至37℃。
    11. 在冰上解冻Matrigel。
    12. 离心管,胶原酶溶液在520 xg下于25℃下10分钟。
    13. 使用连接到真空抽吸的巴斯德玻璃移液管吸出上清液,留下约1ml的介质在颗粒的顶部。
    14. 重要提示:除非另有说明,否则所有塑料移液器和管道应在使用前立即用BSA溶液预先涂覆,以避免将有机物粘到塑料表面。
    15. 使用预先包被的移液管吸头将颗粒重悬于10ml预热的DMEM / F12培养基中。
    16. 将悬浮液转移到已经用BSA溶液预包被的15ml离心管中
    17. 离心管在520 xg下于25℃下10分钟。
    18. 如步骤B13所述吸出上清液。巴斯德移液器不需要在使用前用BSA预涂。
    19. 使用预先包被的10ml移液管吸头,将沉淀重悬于4ml DNase溶液(配方3)中。
    20. 用手轻轻晃动2-5分钟。
    21. 加入6ml的DMEM / F12,并用预先包被的10ml移液管吸头彻底重新悬浮。
    22. 通过细胞过滤器将悬浮液过滤到新鲜的预包被的15 ml管中,以去除胶原酶消化后残留的大块。
    23. 离心管在520 xg下于25℃下10分钟。
    24. 如步骤B13所述吸出上清液。巴斯德移液器不需要在使用前用BSA预涂。
    25. 使用预先包被的移液管尖端将沉淀重悬于10ml的DMEM / F12培养基中
    26. 脉冲离心管在25℃下加热至520×g。一旦达到520 x g,就停止离心机。
    27. 重复步骤B25-B27三次(共四次),或直到脉冲离心后的培养基清晰。最后一步,小心删除整个介质。
    28. 使用预先包被的移液管尖端将沉淀重悬于10ml的DMEM / F12培养基中
    29. 确保悬浮液混合均匀。使用微量移液管取出50μl,转移到无菌的5cm细胞培养皿中,以确定溶液中有机物的密度。
    30. 在显微镜下计数50μl的有机物数量。有机物被计入盘中,不需要盖玻片。准备后立即乳腺肿瘤组织的实例如图1所示。根据肿瘤的大小,每个制剂的总有机体含量可以从约5,000到40,000不等。当前实验不需要的有机体可以如步骤B42所述进行冻结和储存。


      图1.有机体的分子形态发生。在该实施例中,从MMTV-PyMT衍生的乳腺肿瘤制备组织细胞。左图:有机物在第0天,组织准备后,嵌入Matrigel。右图:在第6天的有机物,在FGF碱基存在下生长,嵌入Matrigel。

    31. 离心管在520℃下在25℃下2分钟。
    32. 使用巴斯德玻璃移液器,将上清液完全小心地吸出。巴斯德移液器不需要在使用前用BSA预涂。
    33. 根据以下标准将Matrigel中的有机体颗粒重悬,立即放入冰中。由于Matrigel的高蛋白质含量,移液器吸头和细胞培养板不需要用BSA预包被该步骤。为了在24孔细胞培养板中培养有机物,需要每孔80μl。有机体电镀密度根据下游测定而变化。对于显微镜分析,板组织细胞密度为2个有机体/μl(见注2)。对于RNA提取,板组织细胞的密度为5个有机体/μl。一个井产生足够量的RNA以进行下游的qRT-PCR测定;如果需要更高的RNA产量,则在提取RNA时将两个或更多个孔进行接种(参见方法C:从有机体中提取RNA)。
    34. 准备无菌预热(37℃)的24孔细胞培养板,并将其置于装有无菌水的预温热(37℃)75cm 2 / sup细胞培养瓶的顶部。充满水的烧瓶像加热板一样起作用,对Matrigel的快速固化至关重要
    35. 小心地将含有基质胶的有机物质平铺在孔中部的80μl圆顶(见注1)。避免气泡。移液管吸头不需要用BSA进行预涂,可用于此步骤。
    36. 将培养板在细胞培养箱中孵育5%CO 2和37℃20分钟。
    37. 每孔加入1ml预热的(RT)有机介质(Recipe 4)。小心不要打扰Matrigel圆顶。
    38. 在无菌条件下向培养基中加入反义寡核苷酸(ASO)。单独添加ASO,每井一个。最佳ASO浓度根据ASO和靶基因而变化。应执行0.1-5μM范围内的滴定(见注3)。作为起点,每孔5μM的浓度(25μl,200μMASO溶液)应该导致> 50%的大多数目标击倒。 ASOs由乳腺组织自由摄取,不需要转染试剂。
    39. 至少留下一口井未经治疗,并用至少一口井将ASO(scASO)作为阴性对照。
    40. 在5%CO 2和37℃的细胞培养箱中孵育有机物。
    41. 将培养基更换为新鲜的有机体培养基,并在组织发生后三天补充ASO。有机体应在产后6天内收获(见附注4)。在FGF存在下培养6天的乳腺组织细胞的一个例子如图1所示
    42. 可选:当前实验不需要任何额外的有机物,可以进行冷冻和储存。为了这样做,在步骤B30计算所需的有机物的量和有机体悬浮液的相应体积。将有机体悬浮液分成两部分,一个含有实验所需的量,另一个含有任何其他有机物。如步骤B31和B32所述进行两个等分试样。重新悬浮Matrigel实验所需的有机物,如上所述进行。将额外的有机物重新悬浮在冷冻介质(配方5)中,等分在冷冻机中,并转移到CryoSafe冷冻控制器。在-80℃下保存过夜,并在第二天转移到液体N 2。为了解冻有机体,将小瓶温至37℃,并将组织重悬于10ml的DMEM中。按照步骤B31至B37对Matrigel中的有机物进行平板化。对于解冻的有机物,推荐使用5-10种有机物/μl的种子密度,因为并非所有的有机体都能在冻融过程中生存。

  3. 从有机体中提取RNA
    1. 可以在ASO加入后24小时收获有机物,以测定击倒效率。如果需要进行表型分析,可以在ASO加入后第3天6天(有机体制剂的第0天)进行活细胞成像,因为分支形态发生在3-4天左右开始。或者,可以在第6天进行端点显微镜分析,以比较ASO处理的有机体以控制有机体。在这种情况下,仍然可以在第6天测定击倒效率(见注4)
    2. 将含有有机物的24孔板从细胞培养箱转移到冰箱(4℃)。这一步将导致Matrigel过夜液化。
    3. 第二天,将每个孔(培养基和Matrigel)的内容物转移到冰上的15ml离心管中。
    4. 向每个孔中加入200μl冷细胞回收溶液以解聚任何残留的Matrigel,并在4℃下孵育1小时。
    5. 转移每个孔(细胞回收溶液和Matrigel)的内容物,并结合以前去除的有机体/ Matrigel混合物。如果Matrigel粘在底部,可以使用仔细的刮擦来分离Matrigel和有机物
    6. 在冰上孵育1小时。
    7. 在4℃下以420g离心10分钟。
    8. 使用微量移液管仔细吸取上清液。
    9. 将有机体沉淀重悬于100μl冷细胞回收溶液中
    10. 在冰上孵育10分钟。
    11. 在4℃下以420g离心5分钟。
    12. 使用微量移液管小心吸取上清液。
    13. 将沉淀重悬于1ml 1x DPBS(不含RNase)中。
    14. 在4℃下以420g离心5分钟。
    15. 使用微量移液管小心吸取上清液。
    16. 将细胞沉淀重悬于1ml TRIzol中
    17. 根据制造商的说明从有机体分离RNA。推荐添加糖原(如GlycoBlue),因为如果有机体以低密度接种并在24小时后收获,RNA产量可能很低。将RNA沉淀重悬于10-20μl无核酸酶的水中
  4. cDNA合成和qPCR分析击倒效率
    1. 使用NanoDrop 2000 UV-Vis分光光度计测量RNA浓度。始终保持RNA在冰上,以免降解。
    2. 使用总共1,000 ng RNA,体积为8μl(浓度为125 ng RNA /μl)。如果浓度较高,则在无核酸酶的水中稀释。
    3. 将样品转移到0.2 ml PCR管中
    4. 通过向RNA样品中加入1μl10×DNase反应缓冲液和1μlDNA酶I(总体积:10μl),进行DNA酶消化去除任何潜在的共同纯化的基因组DNA。
    5. 通过轻轻移液上下混合反应。在离心机中短暂旋转。
    6. 在PCR机中在25℃下孵育反应15分钟
    7. 加入1μl25 mM EDTA
    8. 通过轻轻移液上下混合反应。在离心机中短暂旋转。
    9. 在PCR循环仪中在65℃下反应10分钟
    10. 在10分钟孵育完成后立即将样品转移到冰上。不要将样品放在PCR机中,同时倾斜到RT。
    11. 准备cDNA主混合物(配方6)。
    12. 在冰上将样品加入39μlcDNA Master混合物
    13. 通过轻轻移液上下混合反应。在离心机中短暂旋转。
    14. 在PCR机器中进行以下孵育:
      25°C 10分钟
      48°C 30分钟
      95°C 5分钟
    15. cDNA可以短期储存在4°C或长期保存在-20°C
    16. 准备包含靶基因引物的qPCR主混合物(配方7)(见附注5)以及含有内部对照(“家务”)基因引物的qPCR主混合物。在计算需要的主混合料量时,应考虑将样品一式三份移至。
    17. 常用的内部控制基因包括β-肌动蛋白,GAPDH或核糖体蛋白基因。应仔细选择内部控制;其表达水平应与实验条件(如ASO治疗)一致和独立。内控基因的表达水平应与靶基因相同。关于qPCR实验和qPCR引物设计的一般规则可以在(Bustin等人,2009)中找到。
    18. 在冰上准备96孔板。
    19. 将2μlcDNA移入96孔板的一个孔中。每个样品将被移液一次三次,用于目标和持家基因(=每个样品含有2μlcDNA的6个孔)。
    20. 将含有靶基因或内部对照基因引物的18μl主混合物加入孔中
    21. 用光学胶粘剂膜密封96孔板。避免用手指触摸胶片,边缘小心处理。
    22. 在室温下将板旋转100分钟1分钟。
    23. 将板放在qPCR机器中,并运行以下程序:
      95℃10分钟
      40个循环:95℃15秒 60°C 60秒
      其次是
      熔体曲线95°C→60°C,1°C / min

数据分析

  1. 从qPCR机器导出Ct值,并使用2 -ΔΔCt方法(Livak和Schmittgen,2001)进行分析。与未经处理的样品相比,用ASO处理的样品中的靶基因应显着降低,而用scASO处理的样品作为阴性对照。 scASO不应对基因表达产生任何影响,并导致与未处理样品非常相似的值。非常有效的ASO可以产生> 90%的击倒效率,但击倒效率在不同的ASO和目标基因之间变化。应对至少三个独立的生物复制进行统计学上有意义的结果。通过增加使用的ASO的量,测试针对RNA的不同区域的ASOs和不同的有机体密度,可以提高不足的击倒效率。此外,电镀有机物时,ASO可以与Matrigel混合,并添加到介质中以获得最大的击倒效率。
  2. 可以使用标准光学显微镜进行有机体表型变化的端点分析。当比较ASO处理的有机体以控制有机体时,经常观察到数量,大小和分支形态发生的变化。为了进行统计学显着的比较,每次治疗应至少计算100个组织细胞,最少进行3次实验重复。

笔记

  1. 有机物也可以在48孔和96孔板中生长,用于需要更高产量的测定。 Matrigel穹顶的尺寸必须相应地缩小,例如,在48孔板中40μl/孔,在96孔板中20μl/孔。
  2. 如果要进行活细胞成像以分析有机体,请使用玻璃底板(例如,,MatTek)
  3. 如果使用新的未经测试的ASO,建议测试几种浓度(例如,,0.1,0.5,1和5μM),并比较击倒效率。
  4. 根据我们的RNA-seq数据,在人造细胞外基质(ECM)中培养长达七天的有机物不会显着改变lncRNA的表达模式(Diermeier等人,2016)。 br />
  5. 为了获得最佳结果,qPCR的引物不应与ASO结合位点重叠,并且ASO不应在qPCR扩增区内结合。

食谱

  1. 胶原酶溶液(50ml,足以用于两个肿瘤)
    2 ml FBS
    2.5μl庆大霉素(50mg / ml)
    25μl胰岛素 4毫升胰蛋白酶
    75毫克胶原酶
    加入DMEM / F12至50 ml
    无菌过滤器使用前
    准备后立即使用
  2. BSA溶液(2.5%)
    将2.5mg BSA溶于100ml DPBS中 无菌过滤器使用前
    可以重新使用并在4℃下储存长达四周
  3. DNase解决方案
    从牛胰腺(1U /μl)中加入40μlDNase I至10ml DMEM / F12培养基 准备后立即使用
  4. 有机体培养基(10 ml,足够10孔) 100μlPen / strep
    100μlITS
    3μlFGF-basic
    加入DMEM / F12至10 ml
    可以在4°C储存一周
    没有添加FGF2,有机介质可以在4℃下储存长达四周
  5. 冷冻介质
    FBS中的10%DMSO 可以长期保存在-20°C
  6. cDNA主混合物(除了不含核酸酶的H 2 O 2之外的所有组分包括在TaqMan反转录试剂盒中)
    10x TaqMan RT缓冲区
    5μl
    随机六进制文件
    2.5μl
    MgCl 2(25mM)
    11μl
    dNTPs
    10μl
    核糖核酸酶抑制剂  
    1μl
    RTase
    1.25μl
    不含Nuclease的H 2 O /或
    8.25μl
    总量
    39μl
    准备后立即使用
  7. qPCR主组合
    PowerUp SYBR绿色主混音
    10μl
    不含Nuclease的H 2<             
    7μl
    总计
    17μl
    底漆混合(用于+ rev,10μM)
    1 /孔(浓度:0.5μM/孔)
    准备后立即使用

致谢

我们的肿瘤组织细胞方案基于以前的工作形式Andrew Ewald的实验室(Ewald,2013; Nguyen-Ngoc等人,2015)。 Manhasset妇女乳腺癌联盟(S.D.D.)和NCI 5P01CA013106-Project 3(D.L.S.)支持这项研究。

参考

  1. Arun,G.,Diermeier,S.,Akerman,M.,Chang,KC,Wilkinson,JE,Hearn,S.,Kim,Y.,MacLeod,AR,Krainer,AR,Norton,L.,Brogi, ,Egeblad,M。和Spector,DL(2016)。 Genesis Dev 30(1):34-51。
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  5. Clevers,H.(2016)。建模发展与疾病有机体。 细胞 165(7):1586-1597。
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  9. Geary,R. S.,Norris,D.,Yu,R.and Bennett,C.F。(2015)。 反义寡核苷酸的药代动力学,生物分布和细胞摄取。 Adv Drug Deliv Rev 87:46-51。
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引用:Diermeier, S. and Spector, D. L. (2017). Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids. Bio-protocol 7(16): e2511. DOI: 10.21769/BioProtoc.2511.
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