Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system.

K.O. Hartley, S.L. Nutt and E. Amaya

Proc. Natl. Acad. Sci. (USA) 99:1377-1382 (2002) Full Article (1400K)

Abstract

The transgenic technique in Xenopus allows one to missexpress genes in a temporally and spatially controlled manner.

However this system suffers from two experimental limitations. Firstly, the restriction enzyme mediated intergration

procedure relies upon chromosonal damage, resulting in a percentage of embryos failing to develop normally.

Secondly, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in

variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS

method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines, carrying

'activator' or 'effector' constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a

desired promoter, whereas effector lines contain DNA-binding motifs for Gal4 (UAS) linked to the gene of interest.

We show that upon inter-crossing of these lines, the effector gene is transcribed in the temporal and spatial manner of

the activator's promoter. Furthermore we use the Gal4/UAS system to missexpress Xvent-2, a transcriptional target of

BMP4 signalling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector

transgenes display a consistent microcephalic phenotype. Finally we exploit this system to characterise the neural and

mesodermal defects obtained from early missexpression of Xvent-2. These results emphasise the potential of this

system for the controlled analyses of gene function in Xenopus.

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