Transcriptional activation by recombinant GAL4-VP16 in theXenopus oocyte

Abstract

In addition to measuring transcription in vitro (Gerber et al. 1992) or in transient transfection assays (e.g. Xu et al. 1991), an alternative method to study the function of transcription factors is to inject reporter DNA together with either nuclear extract or factor gene into Xenopus oocytes (Rungger et al. 1990; Theulaz et al. 1988). Here we introduce the GALA-derived assay, which was established in other systems, to study transcriptional activation in the frog oocyte. In our experiment, purified recombinant transactivator fusion protein GAL-VP16(40N) (Seipel et al. 1992; P.Rigoni and W.Schaffner, unpublished) was coinjected with a (3-globin reporter gene into the Xenopus oocytes nucleus (germinal vesicle). The reporter promoter contained five GALA binding sites upstream of an octamer motif. Xenopus oocytes were prepared as described previously (Rungger et al. 1990; Bertrand et al. 1991). Mixtures of 4 ng reporter gene plasmid, 1 ng reference gene and 1 ng purified protein G-AL-VP16(40N) in 10 nl of DNA injection buffer (89 mM NaCl, 1 mM KCl, 20 mM Hepes, pH 7) were injected into the nucleus (germinal vesicle). Following the injections, the oocytes were incubated for 15 to 20 hrs in OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, 5 mM Hepes, 0.5 mM PVP with calcium (Wallace et al. 1973)) containing antibiotics (50 iU of

Different Potential of Cellular and Viral Activators of Transcription Revealed in Oocytes and Early Embryos ofXenopus laevis

“…Accordingly, when we injected DNA template into the Xenopus oocyte, it was first relaxed and then organized into a chromatin structure within 120 min, as measured by supercoil density (data not shown). Chromatin formation prior to the injection of transcription factor did not alter the transcriptional response: When we separately injected the factor 3 hours later than the reporter template, we observed a similar level of transcription to that in coinjection experiments (our unpublished data; see also Xu et al, 1993). So far, however, we do not know whether organization of chromatin is necessary for the transcription effects observed.…”

“…However, unlike the transfection experiments in mammalian HeLa cells (Seipel et a/., 1992), reference gene signals are of limited value in the Xenopus system described here. Ref signals were often reduced by the expression of particular transactivator and/or test genes (see also Xu et a/., 1993). This effect is probably due to intracellular competition for limiting factors.…”

Gal4/UAS Transgenic Tools and Their Application to Zebrafish

“…11 Gal4-VP16 has been widely adopted to express exogenous and endogenous genes of interest at high levels in a number of plant and animal systems. [12][13][14] An additional elegant refinement is the split Gal4 expression system developed for Drosophila. 15 In this technique, transcription is restricted to the intersection of expression from two different promoters, with one regulating the Gal4 DNA binding domain fused with a synthetic leucine zipper and the other regulating a complementary leucine zipper fused to the activation domain of Gal4 or VP16.…”

Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation

“…1A). GAL4-based binary systems controlling transgene expression have been used successfully in different organisms, including Drosophila, zebrafish and Xenopus (Fischer et al, 1988;Grabher and Wittbrodt, 2004;Halpern et al, 2008;Xu et al, 1993). However, since the GAL4 transactivation domain is a potential caspase substrate (van Criekinge et al, 1999), it was exchanged for that of the Herpes simplex virus VP16 gene, which is insensitive to caspase cleavage (van Criekinge et al, 1999).…”