Cartilage Fluorescent Reporter Transgenic Zebrafish
Abstract (Set) In mammalian model organisms, cartilage formation and maturation is visually inaccessible, whether in the fetus or postnatally. Cell culture models for cartilage formation are readily accessible, but cannot exactly mimic in vivo conditions under which cartilage is formed and matures. Because the zebrafish embryo develops externally and rapidly, and is almost transparent, many developmental processes can be directly observed. With the addition of a fluorescent reporter gene, GFP, expressed specifically in the cartilage, JHU scientists have overcome the barriers to direct observation of cartilage during normal development. They have generated a line of transgenic zebrafish that express green fluorescent protein (GFP) in cartilage elements of the developing skeleton, allowing the tissue to be visualized in living organisms through the use of stereomicroscope. The transgene was created by ligating regulatory regions associated with the zebrafish gene encoding a chain of type I collagen to the GFP coding sequence in a vector based on the Tol2 transposable element. The transgene was introduced into the zebrafish genome through embryo injections, and the progeny of injected fish were screened for germ line transmission by inspection for appropriate GFP expression. Description (Set) Proposed Use (Set) The invention as described has immediate utility as a substrate for evaluating compounds thought to have an effect on cartilage formation, growth, or other properties, or for finding new compounds with potential for such actions through a high-throughput screen of a molecular library. Also, the invention can be used as a background for a mutagenesis screen, to uncover genes required for proper cartilage formation or function. The invention can be altered by exchanging the GFP for another coding sequence, and generating transgenic fish expressing another protein in cartilage. Examples would include alternative fluorescent reporters; GAL4 or a derivative of GAL4, to activate expression of a second transgene containing UAS sequences; and CRE recombinase, to allow cartilage-specific recombination in a second transgene containing LoxP sites.
Fisher, Shannon ,
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