Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system.
The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, 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 that carry "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 on intercrossing 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 misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling 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 characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.
Hartley
Katharine O
KO
Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, United Kingdom.
Nutt
Stephen L
SL
Amaya
Enrique
E
D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D003587:Cytomegalovirus / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000502:physiology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D009024:Morphogenesis; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D014982:Xenopus laevis / Q000196:embryology
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors