Dr. Balciunas Zebrafish combine vertebrate biology with many advantageous features of invertebrates, such as external development of optically transparent embryos, small size and relatively simple and inexpensive maintenance. These features, lately enhanced by utilization of transgenic lines marking specific cells or tissues with fluorescent protein expression, enabled mutagenesis screens which propelled the zebrafish into the mainstream of developmental genetics. The success of zebrafish genetics in analyzing biological processes occurring after development is complete- adult physiology and behavior, tissue homeostasis and regeneration- has been more limited. One of key roadblocks in this post-embryonic research has been inability to generate conditional mutants.

My laboratory has taken on the challenge to address this shortcoming by developing fully conditional gene trap vectors for zebrafish. Gene traps contain a reporter flanked by a splice acceptor and a transcriptional termination / polyadenylation signal. In previous work we have found that (i) removal of the AUG codon of the reporter is needed for high stringency of gene traps and (ii) use of fish-derived splice acceptor and polyadenylation sequences is required to achieve high degree of mutagenicity. We have built on this work by modifying gene trap vectors to utilize Gal4-VP16 as the primary reporter. The advantages of Gal4-VP16 as a reporter are increased sensitivity (signal is amplified through Gal4 Upstream Activator Sequence, or UAS) and the possibility to use gene trap lines which do not yield a discernable mutant phenotypes as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS. Our first-generation gene trap vector is flanked by direct loxP sites, making mutants reversible by expression of Cre recombinase. To date, we were able to revert all our mutant alleles using Cre. Our second-generation vectors utilize variants of loxP and FRT sites to make them fully conditional: mutations can be reverted using either Cre of Flp recombinase, and then Flp-reverted alleles can be mutated again using Cre recombinase. We are currently testing the role of one of the first genes mutated with a second-generation vector, tbx5a, in cardiac regeneration.