Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath
Porazinski, S. R., Wang, H., Furutani-Seiki, M. Microinjection of Medaka Embryos for use as a Model Genetic Organism. J. Vis. Exp. (46), e1937, doi:10.3791/1937 (2010).
In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.
1. Mating and collecting eggs
2. Cleaning embryos
3. Making agarose plates for holding embryos during microinjection
Embryos are held in troughs during injection made with a plexiglass mould in 1.5% agarose (Figure 1). The concentration of agarose is important to hold embryos properly.
4. Microinjection of medaka embryos
In medaka, DNA, RNA, morpholino oligonucleotides and tracer dyes are microinjected through the chorion into the cytoplasm of the 1 to 64-cell stage embryo rather than the yolk as in zebrafish. Since the cytoplasm is smaller and less visible in medaka, practice by injecting dyes such as rhodamine-dextran is recommended to develop this skill. Addition of phenol red as a tracer is also encouraged to allow confirmation of injections.
As the chorion surrounding medaka embryos is tough, a short, strong and wide injection needle is required (Figure 2). Prepare this strong wide injection needle from a filament-containing glass capillary.
5. Representative results
Figure 4. Representative images of injected medaka embryos. Panel A shows a representative image of correct injection of rhodamine-dextran into the cytoplasm of a one-cell stage medaka embryo. Panel B shows a representative image of incorrect injection of rhodamine-dextran into the yolk sac of a one-cell stage medaka embryo. Panels C and D show the embryos from panels A and B respectively at approximately 30 hours post-injection. EM: embryo, anterior is upwards in panels C and D and view is dorsal. Note the body of the embryo in panel D is not red as the dye was incorrectly injected into the yolk sac. Arrows in B and D indicate rhodamine-dextran injected incorrectly into the yolk sac note the distinct circular boundary. Dotted lines in A and B indicate outline of cytoplasm of one-cell, view is lateral.
In this video, we demonstrate a method for cell tracer, mRNA, DNA or anti-sense oligonucleotide injection into one-cell stage medaka embryos. This technique has allowed us to study various developmental processes in-vivo in real-time. This process and the subsequent detailed analysis it affords has the potential to significantly enhance our understanding of vertebrate organogenesis and the underlying cell biology. Such knowledge may be important for, and yield therapeutic applications in regenerative medicine.
This work was supported by a grant from the MRC.
|4" fine net||Tools||J & K Aquatics Ltd.||MD001||Used to capture fish during egg harvesting.|
|Embryo medium||Reagent||Home made||200ml 50X stock solution, 1ml 1%methyleneblue in H2O/10 liter RO water (50X stock solution: NaCl 14.7g, KCl 0.6g, CaCl2 2H2O 2.4g, MgSO4 7H2O 4.0g/1 litre RO water.|
|Fine waterproof sandpaper - p2000 grit size||Tools||Hermes Abrasives Ltd.||Very fine grit size for rolling/cleaning embryos with chorions.|
|Borosilicate glass capillaries||Tool||Harvard Apparatus||GC100-10F||For making microinjection needles.|
|Micropipette puller||Equipment||Narishige International||Model PP-830||For making microinjection needles.|
|Eppendorf microloader||Tool||Eppendorf||5242956.003||For loading microinjection needles.|
|Microinjector apparatus||Equipment||Eppendorf||Model 5242.|
|Micromanipulator||Equipment||Narishige International||Model MN151.|
|Agarose injection plate||Tools||Home made||For holding embryos during injection.|