Figure 1.
Generation of CAGs-LSL-rtTA3 and CAGs-LSL-RIK strains.
A. Schematic representation of CAGs-LSL-rtTA3 and CAGs-LSL-rtTA3-IRES-mKate2 (RIK) alleles targeted to the Rosa26 locus, prior to and following Cre-mediated recombination. B. Correctly targeted CAGs-LSL-rtTA3 ESCs (Y1), were retargeted by recombinase mediated cassette exchange (RMCE) to introduce a TRE-GFP-miR30 (TGM) construct to the col1a1 recipient locus. Targeted cells were transduced with adenovirus expressing Cre and plated at low density to isolate individual clones. Clones were treated with doxycycline (1 ug/ml) for 2 days and analyzed by flow cytometry. Graph represents GFP fluorescence of TGM containing, dox-treated CAGs-LSL-rtTA3 (black line) and recombined CAGs-rtTA3 (green line) clones.
Table 1.
Mendelian transmission of Rosa26-targted CAGs-rtTA3 transgenes.
Figure 2.
CAGs-rtTA3 and CAGs-RIK show strong expression in adult tissues.
Whole mount epifluorescence images of small intestine, skin, pancreas kidney and liver from R26-rtTA, CAGs-rtTA3 and CAGs-RIK transgenic animals (all containing TG-Ren.713). R26-rtTA shows strong expression in intestine and skin but weak or patchy expression in most other solid organs. CAGs-rtTA3 and CAGs-RIK show almost identical expression patterns in adult mice. CAGs-RIK mice show strong and consistent expression of mKate2.
Figure 3.
GFP induction and mKate2 expression is uniform in most organs of CAGs-rtTA3 and CAGs-RIK mice.
Immunofluorescence stains for GFP and mKate2 in the small intestine and pancreas of ‘no rtTA’, R26-rtTA, CAGs-rtTA3 and CAGs-RIK mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in small intestine (A), but only CAGs-rtTA3 and CAGs-RIK show robust and uniform GFP expression (and mKate2 for RIK) in the pancreatic acinar tissue (B).
Figure 4.
CAGs-rtTA3 and CAGs-RIK enable GFP induction in myeloid and T lymphocyte lineages.
A. Scatter plots representing GFP and mKate2 expression in Gr1 positive cells in the bone marrow of double transgenic (rtTA/TGM) animals following 1 week of doxycycline treatment (625 mg/kg in chow). B. Quantitation of GFP and mKate2 positive cells in Gr1, Thy1 and CD19 positive populations from the bone marrow, thymus and spleen respectively. Bars represent the mean percentage of GFP or mKate2 positive cells in each tissue, in 3 independent animals (per genotype) +/− SEM. C. Western blot of lysates from control (c57Bl/6), GFP negative and GFP positive splenocytes, indicating rtTA3, GFP and mKate2 expression in each population. Retrovirally transduced 3T3 cells serve as the positive control for rtTA3 expression. D. Graphs represent mRNA abundance in control (C57Bl/6), GFP negative and GFP positive splenocytes.
Figure 5.
Adenoviral Cre induces mosaic activation of rtTA and GFP induction in CAGs-LSL-rtTA3 and CAGs-LSL-RIK animals.
A. Immunofluorescent stains for GFP and mKate2 in liver sections of TG-Ren.713;CAGs-LSL-rtTA3 and TG-Ren.713;CAGs-LSL-RIK mice 1 week following intravenous injection of Adenoviral Cre (5×108 PFU) or PBS (CAGs-LSL-RIK only – left panel) and dox treatment. Double transgenic mice exposed to AdenoCre show mosaic expression of GFP (CAGs-LSL-rtTA3) or GFP and mKate2 (CAGs-LSL-RIK). No GFP of mKate2 expression was observed in animals not exposed to Cre. B. Immunofluorescent stains for GFP and mKate2 in lung sections of triple transgenic mice (CAGs-LSL-rtTA3 or RIK;TG-Ren.713;LSL-KrasG12D). KrasG12D-induced lung adenomas show strong expression of GFP and mKate2. Lowe panel: higher magnification of the lesion. White arrows indicate rare cells that show mKate2, but not GFP expression.
Figure 6.
CAGs-LSL-RIK enables tissue-restricted expression of TRE-transgenes in transgenic models of disease.
A. Whole mount epifluorescence (top panel) and immunofluorescence images from a quadruple transgenic (CAGs-LSL-RIK;TG-Ren.713;LSL-KrasG12D;Pdx1-Cre) animal, showing induction of GFP and mKate2 in both normal acinar tissue and pre-neoplastic, KrasG12D-induced PanIN lesions (top arrow). As observed in AdenoCre treated lungs, some PanIN lesions did not show GFP or mKate2 staining suggesting incomplete LSL excision in a small proportion of cells. B. Immunofluorescent stains for GFP and mKate2 in mammary tissue of CAGs-LSL-RIK;TG-Ren.713;MMTV-Neu;WAP-Cre transgenic mice treated with dox.