Caudal Fin Regeneration in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 303

and identified mutants are subsequently outcrossed and progeny are shotgun se-
quenced to identify heterozygous germline mutations. This approach has been
successfully applied fin regeneration research. As a result, several regeneration
mutants have been identified including Fgf20 and Sly1 (Nechiporuk et al., 2003;
Whitehead et al., 2005). This approach was modified to allow for the identification of
targeted lesions by TILLING, whereby following chemical mutagenesis specific
exons are amplified and sequenced to determine whether a mutation has occurred
(Wienholds et al., 2003). Insertional mutagenesis was also developed as an alternative
to basic chemical mutagenesis in order to obviate the need for positional cloning.
Insertional mutagenesis exploits the ability of retroviruses and transposons to insert
themselves into a host genome (Gaiano et al., 1996). Following mutagenesis of males
by injection of retrovirus or transposon, genomic DNA is isolated from sperm. The
sequences flanking the insertions are cloned and sequenced to identify genes that were
mutagenized. More recently, zinc finger nuclease (ZFN) technology has enabled
targeted gene knockout in zebrafish by exploiting the inherent ability of Cys 2 His 2 zinc
finger protein (ZFP) transcription factors to target specific DNA sequences (Doyon
et al., 2008; Meng et al., 2008). ZFNs are chimeric fusions between ZFPs and the
nonspecific cleavage domain of Fok1 endonuclease. The ZFPs confer exquisite
binding specificity to allow the introduction of double stranded breaks by endonu-
clease activity. Double strand breaks are repaired by error-prone nonhomologous end
joining resulting in the generation of a pool of mutants that contain both insertions and
deletions at the target site. Mutants are identified by amplification and sequencing of the
targeted genomic regions. ZFN technology is advantageous because in addition to
allowing the introduction of specificmutations, germline transmission efficiency is high
where > 10% of progeny carry mutations in genes of interest (Doyon et al., 2008; Meng
et al., 2008).
22.6.2 Transgenics
Transgenesis or the ability to insert foreignDNA into a host genome is used extensively
in fin regeneration studies. This approach is utilized to express particular gene products
in a highly controlledmanner. Tissue-specific or inducible promoters are used to control
the spatiotemporal expression of the transgene. Transgenesis typically employs fluo-
rescently tagged gene products to enable efficient determination of trans-expressing
progeny. Injection of plasmid DNA or bacterial artificial chromosomes (BACs) into the
cytoplasm of a one-cell stage embryo has historically been the most commonly used
method for creating transgenic lines (Amsterdam and Becker, 2005). Subsequently,
site-specific recombination systems such as Cre/loxP and Tol2 transposon-mediated
transgenesis have been successfully used to create transgenic zebrafish lines (Fisher
et al., 2006; Yoshikawa et al., 2008).More recently, a temporally controlled site-specific
Cre-Tol2 transposon method was developed that significantly increases transgenesis
efficiency (Hans et al., 2009). In addition, next-generationZFNs exploitinghomologous
end-joining repair mechanisms will soon be developed to allow for an additional
method for introducing targeted gene knock-ins into the zebrafish genome.