July 1, 2001;
Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis.
Morpholino (MO) based inhibition of translational initiation represents an attractive methodology to eliminate gene function during Xenopus development (Heasman et al., 2000). However, the degree to which a given target protein can be eliminated and the longevity of this effect during embryogenesis has not been documented. To examine the efficacy of MOs, we have used transgenic Xenopus lines that harbour known numbers of integrations of a GFP reporter under the control of the ubiquitous
and highly expressed CMV promoter (Fig. 1a). In addition we have investigated the longevity of the inhibitory effect by using transgenic lines expressing GFP specifically in the lens
of tadpoles. These transgenic lines represent the ideal control for the technique as the promoters are highly expressed and GFP can be easily detected by fluorescence and immunoblotting. Moreover, as GFP has no function in development, the levels of inhibition can be tested in an otherwise normal individual. Here we report that MOs are able to efficiently and specifically inhibit the translation of GFP in transgenic lines from Xenopus laevis and Xenopus tropicalis and the inhibitory effect is long-lived, lasting into the tadpole
stages. genesis 30:110--113, 2001.
[+] show captions
FIG. 1. MO based inhibition of GFP fluorescence in transgenic X. laevis and X. tropicalis. (a) CMV- GFP transgenic X. laevis (top, F2, single integra- tion (Marsh-Armstrong et al., 1999); and X. tropicalis (bottom, F1, multiple integrations, gen- erated as previously described (Kroll and Amaya, 1996) (b) Wild-type embryo injected at the two- cell stage into one blastomere with 10 ng FITC labeled control MO (5ﰈ-CCTCTTACCTCAGTTA- CAATTTATA-3ﰈ; gene tools). (c) CMV-GFP trans- genic embryos injected into one cell at the two-cell stage with 2 ng gfp-MO (5ﰈ-AAGTTCT- TCTCCTTTACTCATGGTG-3ﰈ; gene tools). (d) Dose-dependent inhibition of GFP. Both blas- tomeres at the two-cell stage were injected with gfp-MO to give the indicated total dose (ng). (e) Embryos were injected with 1 ng gfp-MO as in (c) and one individual was followed through- out development. (i) X. laevis CryGFP3 (F2, single integration (Bronchain et al., 1999); and (j) X. tropicalis CryGFP3 (F2, single integration (Offield et al., 2000); transgenic embryos were injected as indicated (ng). +/- presence or absence of transgene.
FIG. 2. Complete inhibition of GFP translation in gfp-MO injected embryos. CMV-GFP embryos were injected with the indicated amount of gfp-MO or control FITC MO (40 ng). Each embryo was injected with half the indicated MO into each blastomere at the two-cell stage. Protein was extracted from pools of 20 individuals (50% transgenic) at the indicated time points and immunoblotted as previously reported (Nutt et al., 2001). Blots were incubated with 1/4,000 rabbit anti-GFP (Abcam) and reprobed with 1/2,000 mouse anti-pan erk (clone 16, Transduction Laboratories) as a loading control.