PhotoMorphs™: A novel light‐activated reagent for controlling gene expression in zebrafish

Abstract

Manipulating gene expression in zebrafish is critical for exploiting the full potential of this vertebrate model organism. Morpholino oligos are the most commonly employed antisense technology for knocking down gene expression. However, morpholinos suffer from a lack of control over the timing and location of knockdown. In this report, we describe a novel lightactivatable knockdown reagent called PhotoMorph™. PhotoMorphs can be generated from existing morpholinos by hybridization with a complementary caging strand containing a photocleavable linkage. The caging strand neutralizes the morpholino activity until irradiation of the PhotoMorph with UV light releases the morpholino. We generated PhotoMorphs to target genes encoding enhanced green fluorescent protein (EGFP), No tail, and E-cadherin to illustrate the utility of this approach. Temporal control of gene expression with PhotoMorphs permitted us to circumvent the early lethal phenotype of E-cadherin knockdown. A splice-blocking PhotoMorph directed to the rheb gene showed light-dependent gene knockdown up to 72 hpf. PhotoMorphs thus offer a new class of laboratory reagents suitable for the spatiotemporal control of gene expression in the zebrafish.

Specification of posterior hypothalamic neurons requires coordinated activities of Fezf2, Otp, Sim1a and Foxb1.2

“…In case of fezf2, this is particularly pertinent because we have shown that fezf2 affects early development of the posterior hypothalamus at 1 dpf. In order to distinguish between early and late effects of fezf2, we designed a PhotoMorph (PM) (Tomasini et al, 2009) specific to fezf2 MO sequences, which led to near complete caging of the fezf2 MO and partial uncaging upon UV exposure at 1 dpf (supplementary material Fig. S15).…”

Cyclic Caged Morpholinos: Conformationally Gated Probes of Embryonic Gene Function

“…For example, peptide nucleic acid (PNA)-2-OMe RNA chimeras that form hairpins have been used to conditionally inhibit gene function, [6] although the photoreleased 2-OMe RNAs may be toxic to embryos. [7] Duplexes composed of the targeting MO and two complementary, tandem oligonucleotides connected by a nitrobenzyl-based linker have also been reported (Figure 1c). [7,9] While these duplexes are easier to prepare than hairpin reagents, they release two potentially cytotoxic oligonucleotides upon photolysis and can be less stable in vivo.…”

“…[7] Duplexes composed of the targeting MO and two complementary, tandem oligonucleotides connected by a nitrobenzyl-based linker have also been reported (Figure 1c). [7,9] While these duplexes are easier to prepare than hairpin reagents, they release two potentially cytotoxic oligonucleotides upon photolysis and can be less stable in vivo. Finally, MOs containing four nitropiperonyloxymethyl-caged thymine bases have been synthesized, bypassing the need for an inhibitory oligomer ( Figure 1d).…”

“…In particular, morpholino-based antisense oligonucleotides (MOs) have been widely used to inhibit gene expression in metazoans that develop ex utero (Figure 1a), [1][2][3][4] and caged versions of these reagents (cMOs) can enable conditional gene silencing through targeted illumination. [5][6][7][8][9] Composed of morpholino-based nucleosides and a phosphorodiamidate backbone, these nuclease-resistant probes are typically injected into zygotes as 25-base oligomers, after which they hybridize to complementary RNAs and disrupt splicing or translation. While cMOs can provide important new insights into embryonic gene function, their utility has been hindered by their empirical design, synthetic complexity, in vivo instability, reliance on complementary inhibitors, and/or use of multiple ** We thank A. Puri and I. Hinkson for their assistance.…”

Chemical Tools for Spatiotemporal Regulation of microRNAs

“…This approach was shown to be effective to control eGFP expression in zebrafish. In the Mayer group, photo-activatable MOs were generated by hybridizing the functional MO sequences with complementary caging strands containing a photocleavablenitrobenzyl linkage [9]. The caging strand masked the morpholino activity until applying the UV irradiation,which released the functional MOs to knockdown different genes in zebrafish.…”