栽培<em>秀丽隐杆线虫</em>在三个维度实验室
Summary
我们提出了一个简单的方法来构建三维线虫培养系统称为NGT-3D和NGB-3D。这些可以被用来研究在更类似于天然线虫栖息比标准2D实验室线虫培养板栖息线虫健身和行为。
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
采用经典的线虫的生长介质板线虫的实验室培养是至关重要的该研究线虫提供了数百个重要的发现。在这里，我们提出新的方法来培养线虫中更准确地反映其三维天然栖息地的环境。尽管其它方法已被用于在3D 13观察线虫 ，这是第一个协议，它允许蠕虫培养在固体三维基质。这里展示的两种方法，NGT-3D和NGB-3D，让科学家问3D养身健体，发展壮大的问题，也是图像…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由支持的新研究员格兰特[2014R1A1A1005553]韩国国家研究基金会（NRF）为JIL;和延世大学未来之星挑战格兰特[2015-22-0133]为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 |
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