Morphant technology in model developmental systems

Morpholino phosphorodiamidate oligonucleotides (morpholinos; MOs) are synthetic DNA analogues with some highly favorable properties as in vivo gene targeting tools (Summerton, 1999). MOs have a morpholine ring in lieu of the standard ribose sugar moiety and contain a neutral charge backbone (Fig. 1a). With high affinity for RNA, MOs serve as excellent antisense reagents, although they do not recruit RNAseH, and thus efficacy is achieved through nonclassical antisense approaches. MOs also display little toxicity and have no known cellular binding proteins or nucleases (reviewed in Summerton, 1999). MOs were developed as potential therapeutics (Arora et al., 2000; Qin et al., 2000) and are now being used for functional genomics applications (see Ekker, 2000 for review). The alteration of transcriptional processing of newly synthesized (i.e., zygotic) transcripts is one approach for MO-based gene targeting (Fig. 1b, c). Binding of an antisense MO to one potential intron/exon junction can cause the splicing machinery to effectively skip an entire exon. This strategy, pioneered by Kole and colleagues for therapeutic applications, can be used for the activation of genes if the skipped exon contains a missense or nonsense mutation (Schmajuk et al., 1999). More recent work by Draper and colleagues (2001) has shown its effectiveness for gene inactivation in zebrafish embryos. This method has one significant advantage because the effectiveness at splice alteration can be readily monitored using reverse transcriptase polymerase chain reaction or other, standard RNA analysis techniques. Once the genomic sequence of the zebrafish and other relevant developmental biology systems becomes available (Duyk and Schmitt, 2001), this targeting strategy should become much more amenable to broader use. Translational inhibition is another successful major MO gene targeting strategy (Fig. 2). In this approach, an antisense MO selected against the leader sequence or nearby bases can bind and sterically inhibit scanning of the mRNA by the 40S ribosomal subunit. The resulting double-stranded RNA:MO complex may mimic doublestranded regulatory sites often found in 59 untranslated regions (UTR) of mRNAs. MOs have been shown to invade even native secondary structure due to MO’s high affinity for RNA (Summerton, 1999). Efficacy appears restricted to target sites within the leader and sequences surrounding the start codon (Fig. 2c; Summerton, 1999); a bound MO does not appear capable of altering activity of the ribosomal complex once translation has initiated. Thus, only a small fraction of the transcribed RNA sequences bound by MOs within a cell will result in a deleterious effect on gene function (Summerton, 1999). This mechanism of targeting has been successfully used and published in a variety of systems for gene “knockdown” studies (Table 1). For zebrafish and sea urchin embryos, this is the first viable sequence-specific gene inactivation method. MOs are also an effective tool for Xenopus, chicken and Drosophila melanogaster embryos (Table 1; N. Patel, personal communication; Fig. 3d). The potential impact of this technology is perhaps greatest in systems not amenable to standard genetic approaches. MOs readily function against both zygotic and maternal transcripts for translational inhibition (Table 1). Work with a novel member of the BMP receptor gene family, Alk8 (Bauer et al., 2001), demonstrates one advantage of MO targeting for assigning gene function over conventional zygotic screens. The loss-of-function, zygotic null mutation of Alk8 shows a significantly weaker phenotype than a loss-of-function of BMP ligands because of the translation of functional protein from maternal message pools. In contrast, targeting Alk8 using MOs inhibited translation of both maternal and zygotic message pools, yielding a loss-of-function phenotype of the receptor that was indistinguishable from that of loss-of-function of the ligand. MO targeting has the potential to target a larger pool of genes required for early embryogenesis than would readily be found using conventional zygotic mutagenesis techniques.