Transposon-Generated GFP Gene Traps in Zebrafish

Anna Cristina Garza¹, Stephen L. Johnson², Biology Department, Washington University, St. Louis, MO¹, Department of Genetics, Washington University School of Medicine².

The zebrafish Danio rerio has proven to be a valuable model system in the study of vertebrate developmental biology. Recently, the study of zebrafish has been enhanced by the ability to randomly integrate green fluorescent protein (GFP) into the zebrafish genome. These transgenic fish will improve our forthcoming understanding of developmental genetics by providing us with new observational tools as well as with molecular handles on genes important for various developmental processes.

Zebrafish are an ideal vertebrate model system for a number of reasons. They are small, easily bred, and easily maintained. Furthermore, zebrafish exhibit external fertilization, and thus offspring are readily accessible and observable during embryonic and larval development. Most importantly, zebrafish are transparent until adulthood, allowing for the observation and analysis of the development of individual cells and organs. Therefore, zebrafish are an excellent vertebrate system for our transposon-based GFP screen.

Transgenesis of zebrafish was conducted by means of a transposon, a mobile DNA sequence discovered in maize by Barbara McClintock. The Tol2 element from the medaka fish Oryzias latipes is a transposon used as a gene trap vector in our research; it contains GFP from the jellyfish Aequorea victoria and is inserted randomly into the DNA of a zebrafish to generate transgenic lines expressing GFP. The Tol2 element is similar to Activator (Ac), a transposable in maize, in that it is autonomous and codes for its own transposase; it is, however, rendered nonautonomous in our gene trap construct pT2KSAG by the insertion of GFP. Zebrafish embryos have been coinjected with pT2KSAG and the mRNA coding for Tol2’s transposase, resulting in the transposon's random yet permanent incorporation into the genome. Consequent GFP expression is visible under ultraviolet (UV) light.

These founder fish (the F0 generation) were screened for fluorescent, GFP-expressing cells under UV microscope and sorted into GFP-positive and GFP-negative individuals. (Those expressing GFP in other places are more likely to have the Tol2 element inserted into their germ line as well.) To create stable transgenic lines, GFP-positive males were bred with wild-type females; the F₁ generation was then carefully screened for GFP expression under UV microscope at several developmental stages: 24 hours post fertilization (hpf), 48 hpf, 72 hpf, 1 week post-fertilization (wpf), 2 wpf, and 4 wpf. We have identified several GFP expression patterns; fluorescence has thus far been detected in the heart, trunk, forebrain, midbrain, hindbrain, jaw, developing lateral line system, nasal pits, and caudal and/or pectoral fin buds and/or fins.

The Johnson Lab is currently studying fin regeneration in zebrafish but is, however, limited by observation methods requiring fixed – dead and preserved – tissues. Growth is not a static process and hence requires an observational tool that may be utilized without disrupting normal tissue development; GFP is such a tool. In this particular example, gene-trap transgenic lines of zebrafish expressing GFP in fins – similar to ones we have already identified – would prove invaluable to the study of fin regeneration; one would be able to observe and analyze the division, growth, and development of specific GFP-expressing cells. Once the regulatory elements and genes controlling GFP expression in these cells are identified, new genes involved in fin development and regeneration may be explored.

Similar studies could be conducted in other GFP-labeled lines of zebrafish involving various developing structures. Presently, we are focusing our energy on creating and building reliably distinct and stable stocks of GFP-expressing zebrafish to facilitate this research. GFP lines we create will be valuable tools for studying fin development and regeneration in the Johnson lab and will benefit the zebrafish community overall, as fish with specific expression patterns in various structures will be distributed to interested researchers.