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We have characterized two new transgenic Xenopus lines enabling transgene expression using the Tet-On inducible system. An inducer line expresses the doxycycline- (Dox-) activated transcription factor rtTA under control of the ubiquitous promoter CMV. A responder line enables Dox-inducible expression of a dominant positive thyroid hormone receptor via a tetracycline responsive transgenic promoter (TRE). Dox-induced expression of transgenic GFP mRNA was detectable after 3 hr and increased up to 10- to 50-fold by 2 days depending on dose of Dox. Induced GFP mRNA expression returned to uninduced levels within 3 days upon Dox removal. Treatment of rtTA inducer and TRE responder double transgenic animals with Dox caused acceleration of metamorphic changes in thyroid hormone-response gene expression and morphology. These transgenic lines will be made available through the new Xenopus Stock Center and will serve as valuable tools for genetic analysis of development and metamorphosis.
Figure 1. Chick 5′HS4 insulator sequences improve uniformity of ubiquitous Dox-induced GFP expression. A: Exemplar tadpoles are transgenic for the construct pDPCrtTA-TREG. This construct lacks the HS4 insulators but has the CRY:GFP cassette, as indicated by lens-specific GFP fluorescence. The tadpoles were treated with and without 50 μg/mL Dox for 4 days and visualized using fluorescence microscropy with GFP2 filter sets. The expression of GFP in the body is present but variegated in the presence of Dox and lacking in the absence of Dox. B: Exemplar tadpoles are transgenic for the construct pDPCrtTA-TREG-HS4. This construct has a pair of HS4 direct repeats and also has the CRY:GFP cassette, as indicated by lens-specific GFP fluorescence. The tadpoles were treated with and without 5 μg/mL Dox for 5 days and visualized using fluorescence microscropy with GFP2 filter sets. Again, the expression of GFP in the body is lacking in the absence of Dox, but in this case GFP fluorescence is uniform in the presence of Dox.Download figure to PowerPoint
Figure 2. Dox-induced GFP expression varied across and within founder offspring. Five-day-old tadpoles transgenic for pDPCrtTA-TREG-HS4 were treated with 50 μg/mL Dox for 3 days and shown under brightfield and green fluorescence microscopy. A: All transgenic offspring from each founder lacked visible GFP expression in the absence of Dox, and showed induction of GFP in the presence of Dox. An exemplar tadpole from ♂8 is shown. B: The offspring from four founders (♀1, ♂8, ♂6, ♂1) vary in GFP brightness, likely due to chromosomal insertion site effects. C: A range of Dox-induced GFP expression levels was observed among transgenic sibling offspring from some founders. F1 offspring from ♂8 are shown. A non-transgenic tadpole (left) is shown for comparison.Download figure to PowerPoint
Figure 3. Dose-dependent Dox-induced GFP expression. A,B: Five-day-old offspring from founder ♂8 transgenic for pDPCrtTA-TREG-HS4 were treated with 0, 5, or 50 μg/mL Dox for one day and shown under (A) brightfield and (B) green fluorescence microscopy. In the absence of Dox, GFP is not visible in the body. In the presence of Dox, GFP is induced to a higher level with 50 compared to 5 μg/mL. A representative tadpole from the highest brightness class within each Dox treatment is shown. C,D: For a time course of dose-dependent GFP mRNA expression, GFP mRNA levels in the tail relative to the housekeeping gene rpL8 were measured (C) at 0, 1, 2, and 3 days of 5 and 50 μg/mL Dox treatment and (D) at 0, 1, 3, 6, and 12 hr of 50 μg/mL Dox treatment in 5- to 8-day-old offspring from founder ♂8 transgenic for pDPCrtTA-TREG-HS4 using real-time reverse transcriptase PCR. Non-transgenic tadpoles were included as controls. C: Sample size was n=3, and no tadpoles were pooled. To avoid variation in the results due to variation in GFP expression among siblings in founder ♂8 (seen in Fig. 2C), tadpoles from the highest brightness class were used for all time points (except Day 0), done by selecting similarly bright individuals from a surplus of Dox-treated tadpoles after 24 hr. D: Because tadoles could not be preselected based on GFP brightness prior to tissue harvesting, we used a sample size of n=3 and pooled 4 tadpoles per sample to decrease the chances of obtaining unrepresentative results, given the expected levels of variability in GFP expression among siblings in the same treatment. Letters above the bars represent significance groups based on ANOVA and Bonferroni post-hoc pairwise comparisons, α = 0.05.Download figure to PowerPoint
Figure 4. Dox-induced dpTR causes metamorphic phenotypes. Double transgenic tadpoles for the two constructs shown were treated with 50 μg/mL Dox beginning at NF stage 49 for 12 days (4 tads/200 mL with changing and feeding every 2nd day). Tadpoles singly transgenic for pDPCrtTA-TREG-HS4 were similarly treated and used as a control. A: GFP expression present all over the body in single (top) and double (bottom) transgenic animals from Dox-activated and rtTA-induced GFP from the TRE:GFP cassette in pDPCrtTA-TREG-HS4 was visualized using fluorescence microscopy. Hindlimbs are indicated by brackets. The small inset at the bottom left of each green fluorescence image is the red fluorescence image cropped around the eye to reveal the absence or presence of the pDRTREdpTR-HS4 transgene. The same camera settings were used in both panels. Representative individuals from n=13 single transgenics and n=7 double transgenics for Dox-treated tadpoles starting at NF49–52. B: The gills in the double transgenic animals (bottom) and the single transgenic tadpole (top) are indicated by brackets. The same camera settings were used in both panels. Representative individuals from n=3 single transgenics and n=2 double transgenics for Dox-treated tadpoles starting at NF49.Download figure to PowerPoint
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