Page 270 - The Toxicology of Fishes

250 The Toxicology of Fishes

A TAG MO
ATG mRNA

Inhibition of translation

B exon 1 exon 2 intron 2 exon 3 pre-mRNA
MO-l1E2 MO-E2l2

Altered splicing
wild–type
1 2 3
transcript
cryptic
1 2 3
splice site
skipped
1 2
exon
retained
1 2 3
intron
FIGURE 5.9 Two mechanisms of gene knock-down by morpholino oligonucleotides (MOs). (A) Inhibition of mRNA by
MO targeted to translational start site. (B) Inhibition of pre-mRNA splicing by MOs targeted to splice donor or splice
acceptor sites. For details, see text and Draper et al. (2001), Ekker (2004), and Nasevicius and Ekker (2000). (A color
version of this figure is available from the first author [mhahn@whoi.edu] upon request.)
spliced transcripts can also be targeted for rapid degradation prior to translation (nonsense-mediated
decay) (Baker and Parker, 2004).
Morpholino-modified oligonucleotides are injected at the one- to eight-cell stage and are distributed
and retained in all cells (Ekker and Larson, 2001). They eliminate or greatly reduce the expression of
the targeted protein, as indicated by expression analysis and by the fact that the phenotype of injected
embryos (morphants) (Ekker, 2000) is in most cases indistinguishable from that of zebrafish null mutants
at that locus (Lele et al., 2001; Nasevicius and Ekker, 2000) or mice bearing a null allele at the orthologous
locus (Topczewska et al., 2001). MO-treated zebrafish have been shown to replicate several human genetic
diseases (Nasevicius and Ekker, 2000). MOs function through at least the first 48 to 96 hours of zebrafish
development, during which somitogenesis and organogenesis occur, and knock-downs lasting longer have
been reported (Nasevicius and Ekker, 2000). Although used primarily in zebrafish, MOs have also been
used successfully in other fish, including trout (Boonanuntanasarn et al., 2002) and lamprey (McCauley
and Bronner-Fraser, 2006). The morpholino approach is proving extremely useful for identifying gene
function during development, and it can be accomplished more quickly and with less cost than targeted
disruption of murine loci. Similarly, gene knock-downs are finding application in developmental toxi-
cology (Carney et al., 2004; Incardona et al., 2005, 2006; Linney et al., 2004b; Prasch et al., 2003). For
example, studies using MOs have shown that AhR2 but not AhR1A and ARNT1 but not ARNT2 are
required for TCDD developmental toxicity in zebrafish (Prasch et al., 2003, 2004, 2006).
Another approach for gene targeting in zebrafish is target-selected inactivation, in which zebrafish
generated using mutagenized sperm are screened for point mutations that result in null alleles at specific
loci. In contrast to mouse knock-outs, in which genes are targeted for homologous recombination, target-
selected inactivation involves random mutagenesis followed by screening a large number of individuals
for the desired mutation. The screening process is accomplished by high-throughput resequencing or by
a method known as TILLING (Amsterdam and Hopkins, 2006; Wienholds et al., 2002, 2003) that
facilitates the identification of mutated alleles. Although the use of target-selected inactivation in toxi-
cological research has not yet been reported, this method holds great promise as a complement to MO-
based knock-down approaches.
Gene knock-outs in zebrafish also have been generated by insertional mutagenesis, in which a retrovirus
is used to disrupt genes at random (Amsterdam et al., 1999). The mutated gene is easily identified using
viral sequences as probes. A good example of how such mutants might be used was provided by recent
studies. By taking advantage of an ARNT2 mutant that was one of many mutants generated by random
insertional mutagenesis (Golling et al., 2002), Prasch et al. (2004) were able to establish that ARNT2
is not required for the developmental toxicity of TCDD in zebrafish.