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The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature . Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues .
To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.
In this protocol, we will use the jjxy symbol to denote a hypothetical zebrafish lens mutant.
Fig. 1 Low magnification image of a sharpened needle used for lens transplantation. A glass capillary (B) is inserted into the Pasteur pipette (A), and a thin tungsten wire (C) is inserted into the open end of the capillary. The glass over the wire is softened with a Bunsen burner. Using forceps, hold steady the glass end with the metal wire while twisting the other end of the Pasteur pipette with your hand. The softened glass should spiral around the metal, gripping it in place.
Fig. 2 The tips of the two kinds of needles used for lens transplantation.
Fig. 3 Embryos underwent lens transplantation at 30hpf. Shown are a donor embryo (A, B), a host eye on with transplanted lens (C, D), and the control side of the host embryo (E, F). Each eye is shown in both transmitted light and UV illumination as indicated. Photographs were taken at 48 hpf. The Q01 transgenic line was used in this experiment.
Several steps require special attention during lens transplantations.
This procedure follows to a large extent the transplantation technique developed for cave fish by the lab of Bill Jeffrey.