使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程
Summary
我们描述了分步说明:1) 使用磁性纳米粒子对慢透或抗逆转录病毒转导进行高效工程肠道有机体,以及 2) 从工程有机物生成冷冻部分。这种方法提供了一个强大的工具,有效地改变基因表达在有机体的研究下游效应。
Abstract
肠道器官培养为研究肠道干细胞和体外密码生物学提供了独特的机会,尽管操纵器官基因表达的有效方法在这一领域进展有限。虽然CRISPR/Cas9技术允许对细胞进行精确的基因组编辑,以便产生有机体,但这一策略需要通过序列分析进行广泛的选择和筛选,这既耗时又昂贵。在这里,我们提供了一个详细的方案,为有效的病毒转导肠道器官。这种方法快速、高效,从而减少了CRISPR/Cas9技术固有的时间和费用。我们还提出了一个协议,从完整的有机体培养物中生成冷冻部分,以便用免疫组织化学或免疫荧光染色进行进一步分析,可用于确认基因表达或沉默。在成功转导病毒载体进行基因表达或沉默后,可迅速评估肠道干细胞和密码功能。虽然大多数器官研究采用体外检测,但有机体也可以传送给小鼠进行体内功能分析。此外,我们的方法有利于预测对药物的治疗反应,因为目前可用的疗法通常通过调节基因表达或蛋白质功能而不是改变基因组来发挥作用。
Introduction
长时间将小鼠或人类墓穴细胞培养为小肠或结肠的三维 (3D) 有机体的能力提供了重大突破,因为这些培养物显示了肠道上皮在体内的决定性特征1,2,3.从原发墓穴衍生的有机体能够自我更新和自我组织,表现出与其原产组织相似的细胞功能。事实上,有机物不仅概括了体内墓穴的结构组织,而且概括了许多分子特征,为研究正常生物学和疾病状态提供了有用的工具。为了说明,器官研究揭示了参与组织再生1、2、3、4、5以及可增强功能的药物的新型分子途径。在病理设置6,7。
对肠道干细胞的研究特别感兴趣,因为肠道内衬是高度再生的哺乳动物组织之一,每3-5天更新一次,以保护生物体免受细菌、毒素和其他病原体的侵害。肠道流明。肠道干细胞(ISCs)负责这种非凡的再生能力,从而为研究成人干细胞功能1,2提供了独特的范例。在小鼠的沿线追踪实验表明,分离的Lgr5阳性干细胞可以培养出来,在体外产生3D器官或”迷你胃”,在体外它们密切镜像体内的同类。有机体培养也可以从肠道密码细胞分离物中衍生,这些分离物由祖细胞、ISC 和 Paneth 细胞组成,后者构成体内的上皮利基细胞。事实上,来自原发性肠道密码细胞的有机体培养已经演变为一种相对常规的技术,在大多数实验室使用广泛可用的试剂很容易实现。该模型还适用于RNA测序(RNA-Seq)的基因表达定量分析,以及通过质谱法、免疫组织化学或免疫荧光染色2、4、8对蛋白质进行定量分析。此外,功能遗传学可以使用功能增益(基因过度表达或激活突变基因的表达)或功能丧失(基因沉默或功能丧失突变的表达)方法2。
重要的是,标准质粒DNA或多蛋白的病毒转导方案的低效率和高毒性仍然是该领域的一个主要障碍。虽然CRISPR/Cas9技术允许精确的基因组编辑,这种方法需要耗时的选择,然后是序列验证9。在这里,我们提出了一个病毒转导方案,用于初级肠道器官,通过结合到磁性纳米粒子和应用磁场来优化病毒颗粒的传递。对先前协议4、5、10、11、12、13作了关键修改,并提出提高效率的建议。我们还描述了一种从3D母体凝胶(今称为基底膜基质或基质)中培养的完整有机物生成冷冻部分的方法,以便通过免疫组织化学或免疫荧光染色进行进一步分析。
Protocol
Representative Results
Discussion
成人肠道上皮作为有机物的初级培养物为研究干细胞功能、肠道上皮平衡和病理学1、2、3、病理的分子机制提供了强有力的工具。 4.虽然CRISPR/Cas9技术可用于基因工程有机物9,但它受到基于序列分析的广泛筛选和选择所需的广泛筛选和选择的限制。该协议的目的是提供清晰和简洁的说…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院(R01DK102943、R03CA182679、R03CA191621)、马里兰干细胞研究基金(2015-MSCRFE-1759,2017-MSCRFD-3934)、美国肺协会、Allegheny健康网络- 约翰斯的资助。霍普金斯研究基金和霍普金斯消化系统疾病基础研究核心中心。
Materials
| DMEM | Thermo Fisher Scientific | 11965092 | Base medium for 293T cells |
| DMEM/F12+ | Thermo Fisher Scientific | 12634010 | Base medium for organoid culture medium and organoid digestion buffer |
| OPTI-MEM | Thermo Fisher Scientific | 11058021 | Virus plasmids transfection medium |
| Fetal Bovine Serum | Corning | 35-011-CV | Component of virus collection medium and 293T medium |
| Pen/Strep | Thermo Fisher Scientific | 15140122 | Component of organoid culture medium and crypt dissociation buffer |
| PBS (without Ca2+, Mg2+) | Thermo Fisher Scientific | 10010049 | A wash buffer and component of crypt dissociation buffer |
| Mem-NEAA | Thermo Fisher Scientific | 11140050 | Component of organoid culture medium |
| GlutamaxII | Thermo Fisher Scientific | 35050061 | Component of organoid culture medium |
| HEPES | Thermo Fisher Scientific | 15630080 | Component of organoid culture medium |
| EGF | Millipore Sigma | E9644 | Component of organoid culture medium |
| Noggin | Peprotech | 250-38 B | Component of organoid culture medium |
| R-spondin | R&D | 7150-RS-025/CF | Component of organoid culture medium |
| Human recombinant insulin | Millipore Sigma | I9278-5ml | Component of organoid culture medium |
| Nicotinamide | Millipore Sigma | N3376-100G | Component of Transduction medium |
| Wnt3A | R&D | 5036-WN-010 | Component of Transduction medium |
| Y27632 | Millipore Sigma | Y0503-1MG | Component of Transduction medium |
| 0.5M EDTA | Thermo Fisher Scientific | 15575020 | Component of Crypts dissociation buffer |
| DTT (dithiothreitol) | Thermo Fisher Scientific | R0861 | Component of Crypts dissociation buffer |
| Dispase I | Millipore Sigma | D4818-2MG | Component of organoid digestion buffer |
| DNase I | Millipore Sigma | 11284932001 | Component of organoid digestion buffer |
| matrigel(Growth factor reduced) | Corning | 356231 | Used as a matrix to embed organoids |
| Opti-MEM | Thermo Fisher Scientific | 31985070 | Medium for transfection in viral production |
| ViralMag R/L | Oz Biosiences | RL40200 | Magnetic particles of viral transduction |
| Magnetic plate | Oz Biosiences | MF10000 | Magnetic plate to facilitate viral transduction |
| Lipofectamine 2000 | Thermo Fisher Scientific | 11668019 | Transfection agent in viral production |
| Poly-D-Lysine | Millipore Sigma | A-003-E | Coating for plates before seeding 293T cells |
| 4% Formaldehyde Solution | Boster | AR1068 | Solution to fix organoids |
| O.C.T embedding compound | Thermo Fisher Scientific | 4583S | For embedding of the the organoids |
| 5 mL Falcon polystyrene tubes | Corning | 352054 | |
| 50 mL Falcon Tubes | Sarstedt | 62.547.100 | |
| Orbitron rotator II Rocker Shaker | Boekel Scientific | 260250 | |
| Olympus Inverted microscop CK30 | Olympus | CK30 | for scanning and counting crypts |
| Zeiss Axiovert 200 inverted fluorescence | Nikon | Axiovert 200 | for viewing fluorescence in the crypts |
| Amicon Ultra-15 Centrifugal Filter unit with Ultracel-100 membrane | Milipore Sigma | UFC910024 | For concentrating viruses |
| pluriStrainer 20 µm (Cell Strainer) | pluriSelect | SKU 43-50020 | For preparing organoid fragments |
| Falcon Cell Strainer | Fisher Scientific | 352340 | For preparing cyrpts of similar size after crypt isolation |
| Greiner CELLSTAR multiwell culture plates 48 wells (TC treated with lid) | Millipore Sigma | M8937-100EA | ForD2:D37+D16:D37g organoid fragments |
| Animal strain: C57BL/6J | Jackson Lab | #000664 | For organoid culture |
References
- Cheung, E. C., et al. TIGAR is required for efficient intestinal regeneration and tumorigenesis. Dev Cell. 25 (5), 463-477 (2013).
- Xian, L., et al. HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche. Nature Comm. , (2017).
- Sato, T., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 459 (7244), 262-265 (2009).
- Koo, B. K., et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods. 9 (1), 81-83 (2012).
- Kabiri, Z., et al. Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts. Development. 141, 2206-2215 (2014).
- Boj, S. F., et al. Organoid models of human and mouse ductal pancreatic cancer. Cell. 160 (1-2), 324-338 (2015).
- Boj, S. F., et al. Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients. J Vis Exp. (120), (2017).
- Muñoz, J., et al. The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent ‘+4’ cellmarkers. EMBO J. 31 (14), 3079-3091 (2012).
- Schwank, G., et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 13 (6), 653-658 (2014).
- Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. K. A video protocol of retroviral infection in primary intestinal organoid culture. J Vis Exp. (90), e51765 (2014).
- Sato, T., Clevers, H. Primary mouse small intestinal epithelial cell cultures. Methods Mol Biol. 945, 319-328 (2013).
- Ye, Z., Yu, X., Cheng, L. Lentiviral gene transduction of mouse and human stem cells. Methods Mol Biol. 430, 243-253 (2008).
- Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. Collective invasion in breast cancer requires a conserved basal epithelial program. Cell. 155 (7), 1639-1651 (2013).
- Tesfaye, A., et al. The High-Mobility Group A1 gene up-regulates Cyclooxygenase-2 expression in uterine tumorigenesis. Cancer Res. 67 (9), 3998-4004 (2007).
- Haim, H., et al. Synchronized infection of cell cultures by magnetically controlled virus. J. Virol. 79 (1), 622-625 (2005).
- Xue, X., Shah, Y. M. In vitro organoid culture of primary mouse colon tumors. J Vis Exp. (75), e50210 (2013).
