JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Biology

一种用于培养胚胎<em> C。线虫</em>细胞

Published: September 21, 2013
doi:
10.3791/50649
,
1Department of Physiology and Biophysics,University of Miami

Summary

我们在这里描述的修订协议，用于胚胎C.大型文化线虫的细胞。 C.胚胎线虫使用这种方法的细胞在体外培养时，出现分化和概括的基因的表达在细胞中的具体方式。需要直接访问到细胞中，或从其它组织中的特定细胞类型的分离技术可以对C.施加线虫培养细胞。

Abstract

线虫是一种强大的模型系统中，其中的遗传和分子生物学技术是很容易适用的。直到最近，虽然，需要直接访问到细胞和特定的细胞类型的隔离技术，无法在C应用线虫 。这种限制是由于这一事实，即组织内被加压的角质层这是不容易通过与酶和/或洗涤剂处理消化的局限。基于早期的先驱工作，莱尔德布鲁姆，克里斯滕森和同事1开发出一个强大的方法培养C.线虫胚胎干细胞在大规模。鸡蛋是从妊娠成人分离与漂白/ NaOH处理，随后用壳多糖酶处理以除去蛋壳。胚胎细胞，然后通过手动移液解离，并铺板于血清中丰富的介质基片覆盖的玻璃。在隔离牢房的24小时开始改变形态和表达细胞特异性马克分化RS。C.线虫细胞用这种方法培养的存活最多2周体外和已用于电生理，免疫和成像分析以及它们已被分类并用于微阵列分析。

Introduction

秀丽隐杆线虫 （ 线虫 ）是一款功能强大的模式生物研究细胞功能，分化和行为的分子基础。虽然它的基因组，代谢和生物合成途径类似于脊椎动物“，它的遗传和分子可追踪性都远远大于2。在它的优点是它的外形和简洁的解剖，其快速的生命周期（3天在25°C），短寿命（2周）和大量的后代（> 200）。由于其雌雄同体的性质和生命周期短，分子和基因操作是直接在C。线虫 ，包括转基因动物3,4和诸如RNA干扰5基因敲低技术的应用的生成。C.线虫体和蛋壳是透明的。因此，细胞可以容易地可视化在成人和使用标准显微镜的胚胎。在过去的40年里，C。线虫 </em>的社区已经为下创建的宝贵资源线虫的研究，包括收集了大量的突变体，基因敲除和转基因，解剖和发展6,7的详细说明，包括完整的重建神经系统8，和一个完全测序的基因组这是很好的说明，并将提供给整个社会（ www.wormbase.com ）。

尽管有许多优点，一些实验方法已在C挑战线虫 。这些包括需要访问到组织或细胞类型的细胞和分离的质膜的那些。事实上Ç。线虫组织都在其加压流体静骨架，这是不容易通过酶处理或洗涤剂消化密闭。在20世纪90年代末仪Goodman和珍妮特里士满率先为C的电生理记录的方法线虫神经元和肌肉细胞中原位 9,10。虽然这些方法给了我们重要的见解和神经元在体内的肌肉功能，它们是具有挑战性和低吞吐量。替代的方法来研究细胞功能的体内已经制定了，大多尤其是在使用基因编码的钙传感器，如GCamP和卡默莱昂11-13 体内钙成像。这些方法虽然不允许使用的药理学工具，因为它们是应用在完整的活的动物。

在培养C.第一次尝试线虫细胞在体外大规模被他的博士论文14的制备过程中由莱尔德布卢姆。不幸的是，难以与细胞到衬底的粘附性差遇到，不良细胞的分化和存活无法建立此早期协议作为一个强大的细胞培养方法。 1995年，埃德加和他的同事发表的程序由孤立的单个C和文化来研究细胞分化和形态发生。线虫胚胎15。通过用酶处理和手动解离的组合蛋壳的消化获得的胚胎干细胞，继续增殖，产生高达〜500个细胞15。随后，梁先生和同事培养一个小数字卵裂球的研究肠道形态。它们表明，1 体外分离ë卵裂球产生由通过心尖粘着连接16彼此进行交互创建了一个结构类似于肠腔偏振肠细胞。布氏及其同事还报道了培养℃的类似的方法。线虫胚胎细胞在体外 17。

在此基础上的早期作品，克里斯滕森和同事开发出一种强大的协议培养胚胎Ç。线虫细胞在体外 1。他们表明分离出来的C。线虫细胞可以分化成各种细胞类型和保持它们在体内具有的功能，包括细胞特异性标志物的表达。那些在体内具有挑战性的几种技术，可以在隔离C.应用线虫胚胎细胞。这些包括电1,18 19，成像和免疫化学技术20,21，以及通过荧光激活细胞的特定细胞类型分选（FACS）对细胞特异性cDNA文库22,23的结构的隔离。如RNA干扰（RNAi）的基因敲除技术可以对培养C.施加线虫细胞1，用叠氮基糖作为糖蛋白发现的一个工具了一种新的代谢标记方法最近已在体外培养的C.开发线虫细胞24。

总之，细胞培养方法扩展数组Ø可应用到的C. f技术线虫模型，以努力破译基因功能的生物体的情况下。我们在这里描述的协议C.培养线虫在体外 ，这在很大程度上是基于最早由克里斯蒂安森和他的同事1中所述的协议胚胎细胞。

Protocol

星号（*）表示新的或修改的步骤相比，Christensen 等人 1 1。材质设置在细胞培养过程需要大量的鸡蛋从妊娠的成年人中分离的。 C.成长线虫对8P琼脂平板NA22（可通过线虫遗传协会-长城电脑）接种细菌分离大量鸡蛋。在这些板用蛋白胨的量为8倍，通常是用于NGM板的量。较高的蛋白胨浓度更有效地维系着NA22细菌的生长，这出乎OP50-，生?…

Representative Results

线虫培养的细胞分化并表达细胞特异性标志物 Christensen和使用台盼蓝染色的同事证明，> 99％的胚胎C的线虫细胞存活的分离过程。在9日和镀后22，分别为85％和65％，仍健在1。孤立的胚胎C.线虫的细胞必须坚持的衬底，以区分。细胞没有附着形成团块和它是否生存是不明确的。细胞的分化开始2-3小时电镀后并持续至24小时。 24小时内，细胞承?…

Discussion

线虫是一种功能强大的模式生物破译参与发展，行为和衰老的遗传途径。其方便茎主要从难易程度可以遗传操作，并从它的循环寿命短。尽管它的便利性，C.线虫有其局限性。C.线虫细胞是微小的，在加压的角质层，限制了需要直接访问的细胞，例如电生理和药理学技术，或特定类型的细胞的分离，如通过微阵列分析的基因表达图谱方法的应用局限。该三线虫的细胞培?…

Materials

      REAGENTS
Bacto Peptone VWR International Inc. 90000-382  
Difco Agar Granulated VWR International Inc. 90000-784  
Bacto Tryptone VWR International Inc. 90000-284  
Bacto Yeast Extract VWR International Inc. 90000-724  
Leibovitz’s L-15 Medium (1x) Liquid Invitrogen 11415-064  
Fetal Bovine Serum Invitrogen 16140-063  
Penicillin-streptomycin Sigma P4333-100ML  
Chitinase from Streptomyces Griseus Sigma C6137-25UN  
NA22 Escherichia coli Caenorhabditis Genetics Center    
Peanut Lectin Sigma L0881-10MG  
Sucrose Sigma 57903-1KG  
D-(+)Glucose Sigma 67528-1KG  
Ethylene glycol-bis (2-aminoethylether), N,N,N’,N’- tetraacidic acid (EGTA) Sigma E0396-25G  
Hepes Sigma H3375-500G  
Cholesterol      
NaCl Sigma 57653-1KG  
KCl Sigma P9333-500G  
CaCl2 Sigma C1016-500G  
MgCl2 Sigma M8266-100G  
MgSO4 Sigma M2643-500 g  
K2HPO4 Sigma P2222-500G  
KH2PO4 Sigma P9791-500G  
NaOH Sigma S8045-500G  
KOH Sigma P1767-500G  
Ethanol      
Autoclaved distilled H2O      
Bleach      
      EQUIPMENT
101-1000 μl Blue Graduated Pipet Tips USA Scentific 1111-2821  
10 ml Sterilized Pipet Individually Wrapped USA Scentific 1071-0810  
Ergonomic Variable Volume (100-1000 μl) Pipettor with tip ejector VWR International Inc. 89079-974  
Portable Pipet Aid, Drummond VWR International Inc. 53498-103  
Transfer Plastic Pipet Sterile VWR International Inc. 14670-114  
15 ml Conical Tube USA Scentific 1475-1611  
50 ml Conical Tube USA Scentific 1500-1811  
Sterile 18 gauge Needles Becton, Dickinson and Co. 305196  
Sterile 10 ml Syringes Becton, Dickinson and Co. 305482  
Plastic Syringe Filters Corning 0,20 μm pore size Corning 431224  
Acrodic 25 mm Syringe filter w/5 μm versapor Membrane VWR International Inc. 28144-095  
60×15 mm Petri Dish Sterile VWR International Inc. 82050-548  
100×15 mm Petri Dish Sterile VWR International Inc. 82050-912  
12 mm Diameter Glass Coverslips VWR International Inc. 48300-560  
Clear Cell Culture Plates 24 Well Flat Bottom w/lid Thomas scientific 6902A09  
Dumont #5- Fine Forceps Fine Science Tools 11254-20  
Centrifuge 5702 Eppendorf 022629883  
Laminar Flow Hood      
Inverted Microscope with x10 objective      
Ambient air humidified Incubator      
DOWNLOAD MATERIALS LIST

References

  1. Christensen, M., et al. A primary culture system for functional analysis of C. elegans neurons and muscle cells. Neuron. 33, 503-514 (2002).
  2. Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. . C. elegans II. , (1997).
  3. Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Molecular and cellular biology. 5, 3484-3496 (1985).
  4. Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. The EMBO journal. 10, 3959-3970 (1991).
  5. Fire, A., et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391, 806-811 (1998).
  6. Sulston, J. E., White, J. G. Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans. Developmental biology. 78, 577-597 (1980).
  7. Chalfie, M. Caenorhabditis elegans development. Current opinion in cell biology. 1, 1122-1126 (1989).
  8. White, J. G., Southgate, E., Thomson, J. N., Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 314, 1-340 (1986).
  9. Goodman, M. B., Hall, D. H., Avery, L., Lockery, S. R. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron. 20, 763-772 (1998).
  10. Richmond, J. E., Jorgensen, E. M. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nature. 2, 791-797 (1038).
  11. Miyawaki, A., Griesbeck, O., Heim, R., Tsien, R. Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proceedings of the National Academy of Sciences of the United States of America. 96, 2135-2140 (1999).
  12. Kerr, R., et al. Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron. 26, 583-594 (2000).
  13. Nakai, J., Ohkura, M., Imoto, K. A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nature. 19, 137-141 (2001).
  14. Bloom, L. . Genetic and molecular analysis of genes required for axon outgrowth in Caenorhabditis elegans. , (1993).
  15. Edgar, L. G. Blastomere culture and analysis. Methods in cell biology. 48, 303-321 (1995).
  16. Leung, B., Hermann, G. J., Priess, J. R. Organogenesis of the Caenorhabditis elegans intestine. Developmental biology. 216, 114-134 (1999).
  17. Buechner, M., Hall, D. H., Bhatt, H., Hedgecock, E. M. Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Developmental biology. 214, 227-241 (1999).
  18. Suzuki, H., et al. In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation. Neuron. 39, 1005-1017 (2003).
  19. Carvelli, L., McDonald, P. W., Blakely, R. D., Defelice, L. J. Dopamine transporters depolarize neurons by a channel mechanism. Proceedings of the National Academy of Sciences of the United States of America. 101, 16046-16051 (2004).
  20. Bianchi, L., et al. The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation. Nature. 7, 1337-1344 (2004).
  21. Frokjaer-Jensen, C., et al. Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons. Journal of neurobiology. 66, 1125-1139 (2006).
  22. Von Stetina, S. E., et al. Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system. Genome biology. 8, R135 (2007).
  23. Fox, R. M., et al. A gene expression fingerprint of C. elegans embryonic motor neurons. BMC genomics. 6, 42 (2005).
  24. Burnham-Marusich, A. R., et al. Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery. PloS one. 7, e49020 (2012).
  25. Kim, K. J., Yang, Y. J., Kim, J. G. Purification and characterization of chitinase from Streptomyces sp. M-20. Journal of biochemistry and molecular biology. 36, 185-189 (2003).
  26. Chalfie, M., Wolinsky, E. The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans. Nature. 345, 410-416 (1990).
  27. Parpura, V. Voltage-gated calcium channel types in cultured C. elegans CEPsh glial cells. Cell calcium. 50, 98-108 (2011).
  28. Miller, D. M., Niemeyer, C. J. Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input. Development. 121, 2877-2886 (1995).
  29. Lickteig, K. M., et al. Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 21, 2001-2014 (2001).
  30. Zhang, H., et al. UNC119 is required for G protein trafficking in sensory neurons. Nature. 14 (7), (2011).
  31. Maduro, M., Pilgrim, D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics. 141, 977-988 (1995).
  32. Chalfie, M., Sulston, J. Developmental genetics of the mechanosensory neurons of Caenorhabditis elegans. Developmental biology. 82, 358-370 (1981).
  33. Fukushige, T., et al. MEC-12, an alpha-tubulin required for touch sensitivity in C. elegans. Journal of cell science. 112 (Pt. 3), 395-403 (1999).
  34. Driscoll, M., Chalfie, M. The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. Nature. 349, 588-593 (1991).
  35. Xu, K., Tavernarakis, N., Driscoll, M. Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum. Neuron. 31, 957-971 (2001).
  36. Sakmann, B., Neher, E. Patch clamp techniques for studying ionic channels in excitable membranes. Annual review of physiology. 46, 455-472 (1984).
  37. Strange, K., Christensen, M., Morrison, R. Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies. Nature protocols. 2, 1003-1012 (2007).
  38. Goodman, M. B., Lockery, S. R. Pressure polishing: a method for re-shaping patch pipettes during fire polishing. Journal of neuroscience. 100, 13-15 (2000).
  39. Nickell, W. T., Pun, R. Y., Bargmann, C. I., Kleene, S. J. Single ionic channels of two Caenorhabditis elegans chemosensory neurons in native membrane. The Journal of membrane biology. 189, 55-66 (2002).
  40. Ward, A., Liu, J., Feng, Z., Xu, X. Z. Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nature neuroscience. 11, 916-922 (2008).
  41. Parpura, V. Cell culturing of Caenorhabditis elegans glial cells for the assessment of cytosolic Ca(2)(+) dynamics. Methods Mol. Biol. 814 (2), 153-174 (2012).
  42. Zhang, Y., et al. Identification of genes expressed in C. elegans touch receptor neurons. Nature. 418, 331-335 (2002).
  43. Cinar, H., Keles, S., Jin, Y. Expression profiling of GABAergic motor neurons in Caenorhabditis elegans. Current biology : CB. 15, 340-346 (2005).
  44. Bacaj, T., Tevlin, M., Lu, Y., Shaham, S. Glia are essential for sensory organ function in C. elegans. Science. 322, 744-747 (2008).
  45. Colosimo, M. E., et al. Identification of thermosensory and olfactory neuron-specific genes via expression profiling of single neuron types. Current biology : CB. 14, 2245-2251 (1016).
  46. Shih, J. D., Fitzgerald, M. C., Sutherlin, M., Hunter, C. P. The SID-1 double-stranded RNA transporter is not selective for dsRNA length. RNA. 15, 384-390 (2009).
  47. O’Hagan, R., Chalfie, M., Goodman, M. B. The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals. Nature. 8, 43-50 (2005).
  48. Du, H., Gu, G., William, C. M., Chalfie, M. Extracellular proteins needed for C. elegans mechanosensation. Neuron. 16, 183-194 (1996).
  49. Zhang, S., Banerjee, D., Kuhn, J. R. Isolation and culture of larval cells from C. elegans. PloS one. 6, e19505 (2011).

Tags

Embryonic C. Elegans CellsCell CultureChitinaseCell DissociationCell DifferentiationCell MarkersIn Vitro CultureElectrophysiologyImmunochemistryImagingMicroarray Profiling
check_url/50649?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Sangaletti, R., Bianchi, L. A Method for Culturing Embryonic C. elegans Cells. J. Vis. Exp. (79), e50649, doi:10.3791/50649 (2013).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image