Several unique attributes make zebrafish a popular model to study vertebrate development and human diseases (Dooley and Zon, 2000). These include transparency of embryos, rapid embryonic development, small size, short generation time, availability of mutants, genetic and physical maps, and advance of the zebrafish genome. Lately a possibility to generate mutants by site-specific mutagenesis has been added to a list of methods.

Recent evidence of effective transposition of heterologous transposons Tol2 from Oryzias latipes (Kawakami et al., 2000) and Sleeping Beauty from salmonid (Davidson et al., 2003) in the zebrafish genome opened new opportunities for reverse genetics and transgenesis-based applications. Unlike retroviral insertions, the majority of transposon transpositions may occur close to the donor site. Therefore, any transposon insertion in the genome can potentially be used as a donor site (“launching pad”) to produce novel insertions preferentially into the closely linked genes (Smith et al., 1996). Thus, starting from an initially small pool of transposon insertions, a larger number of insertions into the surrounding regions of the genome may be generated. Remobilization can also facilitate identification of the tagged genes and/or their expression patterns. Excision of a transposon from a donor site can restore function of an affected gene associated with rescue of mutant phenotype and generation of revertants used as the supporting evidence that mutant phenotype is caused by the transposon insertion (Grossniklaus et al., 1998).

Insertional elements can carry selectable markers significantly facilitating selection of transgenics and their subsequent genetic analyses. Furthermore, they provide unique opportunities to identify mutations affecting the development of gametes by quantitative analysis of mutant allele transmission to the next generation (Springer et al., 1995). Because the sequence of the insert is known, identification of the gene affected by the insertion is relatively straightforward, compared with positional cloning of N-ethyl-N-nitroso-urea mutants. Loss-of-function mutations of many genes do not produce obvious morphological phenotypes; therefore, they are usually omitted in classic mutant screens. Reverse genetics using insertional mutagenesis will provide valuable material to address this issue.

Yet another advantage of the insertional approach that has been widely used recently in several species is its adaptation for gene and enhancer trapping. Gene trap constructs, for example, should contain multiple splicing acceptor sequences upstream of a promoter-less reporter gene coding sequence to allow gene-reporter fusion upon insertion into the gene in the same orientation. An enhancer trap construct carries a reporter gene with minimal promoter that is induced once it lands under control of a chromosomal enhancer (Bellen, 1999).

Enhancers can activate the transcription at considerable distance in orientation-independent manner; therefore, the approach is the most effective for monitoring gene activity throughout the genome. Because enhancers can activate the reporters outside of genes, the subsequent identification of the regulated gene can be more difficult. However, in many reported cases, enhancer trap lines did reflect the expression pattern of the genes correctly (Perrimon et al., 1991; Bellen, 1999).

We successfully applied the enhancer trap approach in zebrafish using a modified Tol2 element from medaka (Kawakami et al., 2000; Koga et al., 2002; Parinov et al, 2004). This element uses conservative (“cut and paste”) transposition mechanism, i.e. it is excised from the donor site when the whole element moves to the new position. We started by using the cytosolic version of enhanced green fluorescent protein (EGFP, cytGFP) as a reporter. It turned to be a very efficient method of transgenesis in zebrafish, which later on was also adapted to Xenopus. It increased a success of transgenesis 10-20X paving a road to transfering into this field research methodology already developed for Drosophila (Gal4, Cre-Lox, etc). By using this efficient technique of transgenesis and its modification based on a transposition initiated by injection of transposase mRNA into embryos of the stable transgenic parents, several hundreds of cytGFP-expressing stable transgenics were generated and analyzed using a combination of the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and bioimaging. (Garcia-Lecea et al., 2008; Ke et al., 2008; Vasilyev et al., 2009; Kondrychyn et al., 2009; Poon et al., 2010; Winata et al., 2009, 2010) to capitalize on the optical clarity of live zebrafish embryos followed by similar effort using the membrane-tethered version of KillerRed (Bulina et al., 2006) and several dozens of memKR-expressing stable transgenics were made (Teh et al., 2010; Lee et al., 2010).

Several laboratories pefrormed the insertional mutagenesis screens using transposon-based “gene-breaking cassettes” (Sivasubbu et al., 2006; Clark et al., 2011; Sampath et al., unpublished). These mutants could be found in the dedicated on-line database ZFISHBOOK (http://www.zfishbook.org/).

While we tried to present FP expression in transgenics in a same manner as 3D reconstructions of stacks of confocal images due to obvious limitations imposed by a format of this presentation not all elements of expression could be seen, but they all were documented by scanning all images in the stack. Some of anatomical definitions are relatively broad and/or imprecise due to a lack of time and a large number of specimen that we were dealing with.

The original collection of these transgenics kept at the IMCB, Singapore was terminated and here at the IIMCB, Warsaw only a few lines are kept.

Nevertheless, many lines were distributed to the laboratories worldwide and a significant fraction of these lines were deposited to the EZSC in KIT, Karlsruhe.