Das B and Brown DD (2004), Controlling transgene expression to study Xenop...

XB-ART-3775

Proc Natl Acad Sci U S A 2004 Apr 06;10114:4839-42. doi: 10.1073/pnas.0401011101.

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Controlling transgene expression to study Xenopus laevis metamorphosis.

Das B , Brown DD .

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Sperm-mediated transgenesis of Xenopus laevis is the first application of genetic methodology to an amphibian. However, some transgenes are lethal when they are expressed constitutively. To study the influence of these genes on amphibian metamorphosis and to generate F1 progeny from mature transgenic adults, these transgenes must be placed under the control of an inducible system so that they can be activated at specific times in development. We show that two well known binary inducible gene expression systems supplement transgenesis for the study of X. laevis metamorphosis, one system controlled by the progesterone analogue RU-486 and the other controlled by the tetracycline derivative doxycycline. By inducing a dominant negative form of the thyroid hormone receptor under the control of doxycycline specifically in the nervous system we have delimited the developmental periods within which thyroid hormone controls innervation of the developing limb from the spinal cord.

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Species referenced: Xenopus laevis
Genes referenced: actl6a trdn
Lines/Strains:

Xla.Tg(tubb2b:RTTA,TRE:dnthra-GFP)Brown

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References [+] :

Brasier, Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. 1989, Pubmed

	Fig 2. Inducibility and tissue-specific expression of the Tet system in trans- genic Xenopus F0 and F1 tadpoles. Expression of the GFP reporter gene in a 1-week-old F0 transgenic animal before (a) and after (b) 12 h of treatment with 5 ug/ml Dox. (c) Luciferase activity in the tail extracts of 11 different F0 transgenic animals before (solid bar) and after (open bar) induction with 5 ug/ml Dox for 48 h. Tissue-specific expression in 1-week-old tadpoles induced with 5 ug/ml Dox using the NBetaT (d) and cardiac actin (e) promoters driving inducible GFP. [Bars = 500 um (a, b, and e) and 200 um (d).]
	Fig 3. Kinetics of induction in the brain of the TRDN/GFP fusion protein transgene driven by the NBetaT promoter. Dox (50 ug/ml) was added to the rearing water. The same animal before induction (a), after 12 h of Dox induction (b), and 24 h after withdrawal of Dox (c). (The left is anterior in all images.) (Bars = 500 um.)
	TH-induced proliferation of the cells that line the brain ventricle was monitored by anti-PH3 staining (bright spots) in 1-week-old transgenic tad- poles after 4 days of treatment with 10 nM 3,5,3'-triiodothyronine (a), 10 nM 3,5,3'-triiodothyronine plus 50 ug/ml Dox (b), or untreated tadpoles (c). (The left is anterior in all images.) (Bars = 500 um.)