This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.
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Method Article
Generation of Stable Transgenic C. elegans Using Microinjection
In This Article
Summary
Abstract
Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.
Protocol
Expression Plasmid Construction
Two plasmids are required: one for tissue-specific expression of the gene of interest and a second as selectable transformation marker.
Experimental Plasmid
- Select a promoter that is expressed in the tissue/cell type of interest, for example, a nerve or muscle-specific promoter. If one is to express multiple genes within the same tissue, a promoter cassette plasmid in the Gateway system (Invitrogen) can be used. This approach allows the insertion of any gene of interest downstream of the promoter by recombinational cloning 4. Generally, 2-5 kb of upstream sequenc....
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Discussion
When doing this procedure, it is important to remember to:
- inject directly into the center of the gonad
- not inject too much liquid
- work quickly to prevent dessication.
If you run into problems with the needle, such as it is broken too large, it is best to remake a fresh needle, rather than trying to inject with a less than ideal needle.
The generation of transgenic worms has many applications such as:
- identification of mutant genes, in .......
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Acknowledgements
We wish to acknowledge the cooperative spirit of all Caldwell Lab members. Movement disorders research in the lab has been supported by the Bachmann-Strauss Dystonia & Parkinson Foundation, United Parkinson Foundation, American Parkinson Disease Association, Parkinson's Disease Association of Alabama, the Michael J. Fox Foundation for Parkinson's Research, and an Undergraduate Research.
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Materials
| Name | Company | Catalog Number | Comments | |
|---|---|---|---|---|
| Agarose | Ultrapure | Invitrogen | 15510-027 | |
| Halocarbon Oil, Voltalef | Hulle 10S | elfatochem, France | ||
| Coverglass 18x18 mm | Fisher Scientific | 12-548-A | ||
| Coverglass 20x30 mm | Fisher Scientific | 12-548-5A | ||
| Coverglass 22x50 mm | Fisher Scientific | 12-545-E | ||
| 100 ul Capillary | VWR international | 53432-921 | ||
| Glass Capillary for Needles | "Kwik-Fil" | World Precision Instruments, Inc. | 1B100F-4 | |
| Needle Puller | Tool | Narishige International | Model PP-830 | |
| microINJECTOR System | Tritech Research, Inc. | MINJ-1000 | Scope, Stage, Manipulator | |
| Dissecting, with Fluorescence | Microscope | Nikon Instruments | SMZ800 | |
| Dissecting | Microscope | Nikon Instruments | SMZ645 |
References
- Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484-3496 (1985).
- Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. Efficient gene transfer in C.elegans:....
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