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
发布: 2017年08月20日第7卷第16期 DOI: 10.21769/BioProtoc.2511 浏览次数: 11462
评审: Ralph BottcherAnonymous reviewer(s)
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The authors used this protocol in:
Sep 2016
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
- 5 cm sterile cell culture dish (e.g., Corning, Falcon®, catalog number: 353002 )
- Optional: Disposable plastic Cryomold (e.g., Tissue-Tek Cryomold, Electron Microscopy Sciences, catalog number: 62534-25 )
- 15 ml and 50 ml centrifuge tubes (e.g., Crystalgen, catalog number: 23-2265 ; Corning, Falcon®, catalog number: 352098 )
- 24-well cell culture plate (Greiner Bio One International, catalog number: 662160 )
- Cell culture flask, 75 cm2 (e.g., Corning, Falcon®, catalog number: 353136 )
- Pasteur glass pipettes (e.g., Kimble Chase Life Science and Research Products, catalog number: 63A54 )
- Sterile pipette tips (e.g., Corning)
- Cell strainer 70 micron (e.g., Corning, catalog number: 431751 )
- 0.2 ml PCR strip tubes (e.g., Corning, Axygen®, catalog number: PCR-0208-CP-C )
- 96-well reaction plates (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4346907 )
- Optical adhesive film (e.g., Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: 4311971 )
- Mammary tumor-bearing mouse (e.g., MMTV-PyMT (Guy et al., 1992)) with palpable tumors. Optimal tumor size is ~5-10 mm in diameter
- 200 Proof ethyl alcohol (e.g., UltraPure, catalog number: 200CSPTP )
- Sterile water
- Ice
- Matrigel Growth Factor reduced Basement Membrane Matrix, phenol-red free (Corning, catalog number: 356231 )
- Optional: Tissue-Tek O.C.T. compound (Electron Microscopy Sciences, catalog number: 62550-12 )
- Liquid N2
- Cell recovery solution (Corning, catalog number: 354253 )
- 1x DPBS (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
- TRIzol (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596018 )
- GlycoBlue (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9516 )
- DNAse I, amplification grade, for cDNA synthesis (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: 18068015 )
- Ethylenediaminetetraacetate acid disodium salt (EDTA)
- Fetal bovine serum (FBS) (e.g., VWR, product number: 1500-500 )
- Gentamicin 50 mg/ml (e.g., Lonza, catalog number: 17-528Z )
- Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I1882-100MG )
- Trypsin (e.g., Mediatech, catalog number: 25-054-CI )
- Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138-1G )
- Advanced DMEM/F12 (e.g., Thermo Fisher Scientific, GibcoTM, catalog number: 12634010 )
- Bovine serum albumin (BSA) (e.g., Sigma-Aldrich, catalog number: A2153-100G )
- DNase I from bovine pancreas for organoid preparation (e.g., Sigma-Aldrich, catalog number: D4263-1VL )
- Pen/Strep (e.g., Sigma-Aldrich, catalog numbers: PENNA-100MU and S6501-100G )
- ITS liquid media supplement 100x (Sigma-Aldrich, catalog number: I3146-5ML )
- Murine FGF-basic (PeproTech, catalog number: 450-33 )
- Dimethyl sulfoxide (DMSO) (e.g., Sigma-Aldrich, catalog number: D2650-5x10ML )
- Nuclease-free water (e.g., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
- TaqMan Reverse Transcription Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: N8080234 )
- PowerUp SYBR Green Master Mix (Thermo Fischer Scientific, Applied BiosystemsTM, catalog number: A25743 )
- Chloroform, purified (e.g., Avantor Performance Materials, MACRON, catalog number: 4432-10 )
- Isopropanol, molecular biology grade (e.g., Fisher Scientific, catalog number: BP2618500 )
- Collagenase solution (see Recipes)
- BSA solution (see Recipes)
- DNase solution (see Recipes)
- Organoid medium (10 ml, sufficient for 10 wells) (see Recipes)
- Freezing medium (see Recipes)
- cDNA Master mix (see Recipes)
- qPCR Master mix (see Recipes)
Equipment
- Biological safety cabinet (e.g., NuAire)
- 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 )
- Cell culture incubator (e.g., Heracell i Copper CO2 incubator, Thermo Fischer Scientific, Thermo ScientificTM, model: HeracellTM 150i and 240i , catalog number: 50116050)
- Shaker with temperature control (e.g., Thomas Scientific, catalog number: 1222U12)
Manufacturer: Benchmark Scientific, catalog number: H1000-M . - Centrifuge for 15 and 50 ml tubes (e.g., Eppendorf, model: 5804 )
- Centrifuge for 1.5 ml reaction tubes with cooling function (e.g., Eppendorf, model: 5427 R )
- Vacuum suction
- Micro-pipettes (e.g., Gilson, catalog number: F167300 , catalog number for Thomas Scientific: 1222N73)
- Phase-contrast microscope (e.g., Nikon)
- Optional: Cryo-Safe Freeze Controller (e.g., SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F18844-0000 )
- NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 , catalog number: ND-2000)
- PCR machine (e.g., Applied Biosystems Proflex Thermocycler, Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4484073 )
- qPCR machine (e.g., Applied Biosystems StepOne Plus Real-Time PCR system, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4376600 )
- -80 °C freezer (e.g., VWR, catalog number: 10160-728 )
- Water bath (e.g., PolyScience, catalog number: WB10A11B )
- Optional: liquid N2 freezers (e.g., VWR, catalog number: 82017-934 )
- Refrigerator (4 °C)
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
癌症生物学 > 通用技术 > 分子生物学技术 > 反义寡核苷酸
分子生物学 > RNA > qRT-PCR
细胞生物学 > 细胞分离和培养 > 3D细胞培养
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