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 ug/mL Dox for four 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 ug/mL Dox for five 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.

Dox-induced GFP expression varied across and within founder offspring. Five-day old tadpoles transgenic for pDPCrtTA-TREG-HS4 were treated with 50 ug/mL Dox for three 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.

Dose-dependent Dox-induced GFP expression. A and B) Five-day old offspring from founder ♂8 transgenic for pDPCrtTA-TREG-HS4 were treated with 0, 5, or 50 ug/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 ug/mL. A representative tadpole from the highest brightness class within each Dox treatment is shown. C and 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 ug/mL Dox treatment and (D) at 0, 1, 3, 6, and 12 hrs. of 50 ug/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 amongs 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 hours. 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 four 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.

Dox-induced dpTR causes metamorphic phenotypes. Double transgenic tadpoles for the two constructs shown were treated with 50 ug/mL Dox beginning at NF stage 49 for 12 days (4 tads/200mL with changing and feeding every 2nd day). Tadpoles singly transgenic for pDPCrtTA-TREG-HS4 was 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 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.

Dox-induced dpTR caused changes in gene expression. Tadpoles doubly transgenic for the two constructs shown were treated with and without 50 ug/mL Dox beginning at NF stage 52–54 for three days. A tadpole singly transgenic for pDRTREdpTR-HS4 was similarly treated with Dox and used as a control. Tails were harvested for the analysis. Each lane of the gel represents an individual tadpole. The presence of Dox upregulated the transgene dpTR, which in turn induced expression of the two thyroid hormone response genes, thyroid hormone receptor beta (TRβ) and thyroid hormone-induced basic leucine zipper (bZIP). Consistent expression of the control gene ribosomal protein L8 (rpL8) in each lane indicates comparable sample quality.