El cultivo de<em> Caenorhabditis elegans</em> En tres dimensiones en el Laboratorio
Summary
Se presenta un método sencillo de construir sistemas de cultivo de nematodos 3D llamados NGT-3D y NGB-3D. Éstos se pueden utilizar para estudiar la aptitud de los nematodos y los comportamientos en los hábitats que son más similares a los naturales Caenorhabditis elegans hábitats que las placas de cultivo estándar en 2D elegans C. laboratorio.
Abstract
The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.
Introduction
The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5.
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory.
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ.
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.
Protocol
Representative Results
Discussion
El cultivo de laboratorio de C. elegans usando las placas de medio de crecimiento de nematodos clásicos fue crucial para los cientos de descubrimientos importantes que la investigación en C. elegans ha proporcionado. A continuación, presentamos nuevos métodos para cultivar C. elegans en un entorno que refleja con mayor precisión sus hábitats naturales tridimensionales. Aunque otros métodos se han utilizado para observar C. elegans en 3D 13, este es el primer protocol…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Este trabajo fue apoyado por un investigador de Nueva Concesión [2014R1A1A1005553] de la Fundación Nacional de Investigación de Corea (NRF) para JIL; y la Universidad de Yonsei líder futuro desafío de Grant [2015-22-0133] para JIL
Materials
| LB broth, Miller (Luria-Bertani) | Difco | 224620 | |
| Sodium chloride | DAEJUNG | 7548-4400 | 58.44 MW |
| Agar, Granulated | Difco | 214530 | |
| Peptone | Bacto | 211677 | |
| Calcium chloride, dihydrate | Bio Basic | CD0050 | 2*H2O; 147.02 MW |
| Cholesterol | Bio Basic | CD0122 | 386.67 MW |
| Ethyl alcohol | B&J | RP090-1 | 99.99%; 46.07 MW |
| Magnesium sulfate, anhydrous | Bio Basic | MN1988 | 120.37 MW |
| Potassium phosphate, monobasic, anhydrous | Bio Basic | PB0445 | 136.09 MW |
| 2'-Deoxy-5-fluorouridine | Tokyo Chemical Industry | D2235 | 246.19 MW |
| Potassium phosphate, dibasic, anhydrous | Bio Basic | PB0447 | 174.18 MW |
| Multi-Purpose Test Tubes | Stockwell Scientific | ST.8570 | 8 mL |
| Test Tube Closures | Stockwell Scientific | ST.8575 | |
| Cell Culture Flask | SPL Lifescience | 70125 | 25 cm^2 |
| Research Stereo Microscope | Nikon | SMZ18 | |
| High-Definition Color Camera Head | Nikon | DS-Fi2 | |
| PC-Based Control Unit | Nikon | DS-U3 | |
| NIS-Elements Basic Research, Microscope Imaging Software | Nikon | MQS32000 |
References
- Corsi, A. K., Wightman, B., Chalfie, M. A Transparent Window into Biology: A Primer on Caenorhabditis elegans. Génétique. 200 (2), 387-407 (2015).
- Consortium, C. E. S. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science. 282 (5396), 2012-2018 (1998).
- Choi, J. I., Yoon, K. H., Kalichamy, S. S., Yoon, S. S., Lee, J. I. A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans. ISME J. 10 (3), 558-567 (2016).
- Gaertner, B. E., Phillips, P. C. Caenorhabditis elegans as a platform for molecular quantitative genetics and the systems biology of natural variation. Genet Res (Camb). 92 (5-6), 331-348 (2010).
- Cutter, A. D. Caenorhabditis evolution in the wild. Bioessays. 37 (9), 983-995 (2015).
- Brenner, S. The genetics of Caenorhabditis elegans. Génétique. 77 (1), 71-94 (1974).
- Felix, M. A., Braendle, C. The natural history of Caenorhabditis elegans. Curr Biol. 20 (58), R965-R969 (2010).
- Barriere, A., Felix, M. A. High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations. Curr Biol. 15 (13), 1176-1184 (2005).
- Lee, T. Y., Yoon, K. H., Lee, J. I. NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D. Biol Open. 5 (4), 529-534 (2016).
- Lewis, J. A., Flemming, J. T., Epstein, H. F., Shakes, D. C. Basic Culture Methods. Caenorhabditis elegans.: Modern Biological Analysis of an Organism. 48, 4-27 (1995).
- Choi, S. Y., Yoon, K. H., Lee, J. I., Mitchell, R. J. Violacein: Properties and Production of a Versatile Bacterial Pigment. Biomed Res Int. , 465056 (2015).
- Solis, G. M., Petrascheck, M. Measuring Caenorhabditis elegans life span in 96 well microtiter plates. J Vis Exp. (48), (2011).
- Kwon, N., Pyo, J., Lee, S. J., Je, J. H. 3-D worm tracker for freely moving C. elegans. PLoS One. 8 (2), e57484 (2013).
- Pierce-Shimomura, J. T., Chen, B. L., Mun, J. J., Ho, R., Sarkis, R., McIntire, S. L. Genetic analysis of crawling and swimming locomotory patterns in C. elegans. Proc Natl Acad Sci U S A. 105 (52), 20982-20987 (2008).
- Kwon, N., Hwang, A. B., You, Y. J., SJ, V. L., Je, J. H. Dissection of C. elegans behavioral genetics in 3-D environments. Sci Rep. 5, 9564 (2015).
