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. 2005 Mar 22;33(5):e51.
doi: 10.1093/nar/gni051.

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Gusztav Belteki  1 , Jody Haigh, Nikolett Kabacs, Katharina Haigh, Karen Sison, Frank Costantini, Jeff Whitsett, Susan E Quaggin, Andras Nagy
Affiliations

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Gusztav Belteki et al. Nucleic Acids Res. .

Erratum in

  • Nucleic Acids Res. 2005;33(8):2765

Abstract

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.

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Figures

Figure 1
Figure 1
Targeted insertion of a conditional rtTA-IRES-EGFP transgene into the ROSA26 locus. (A) Targeting strategy. The exons of the ROSA26 gene are depicted as numbered boxes, and the triangles as loxP sites. The insertion point (XbaI site), informative restriction sites and diagnostic fragments are also shown. Abbreviations are explained in the text. Drawing is not to scale. (B) Southern-blot analysis of genomic DNA from ES cells digested by EcoRV and hybridized with a 5′ external probe (left) and neo probe (right) (C) After Cre-excision, cells show green fluroescence. Line 1C12 was transfected with a plasmid carrying Cre and puro expression cassettes. A puromycin-resistant colony is shown. (D and E) Tetracyclin-induction of lacZ in Cre-excised 1C12 cells. The cells were transfected with a plasmid containing a tet-O-lacZ transgene. (D) No doxycycline induction. (E) Cells after doxycycline administration (100 ng/ml for 2 days).
Figure 2
Figure 2
Operation of the Cre/loxP-dependent, tetracycline inducible transgenic system. (A) Mice that are homozygous for the targeted insertion of the conditional rtTA-IRES-EGFP bicistronic transgene at ROSA26 can be bred with double transgenic mice that carry a tissue-specific Cre transgene and a doxycycline-inducible rtTA-dependent tet-O-GENE responder line. A total of 25% of the pups will be triple transgenic (SpecPromoterCreTg/+, ROSA26-STOP-rtTA-IRES-EGFPTg/+, tet-O-GENETg/+). (B) In cells that do not express Cre, neither rtTA nor EGFP protein is generated; therefore, the tet-O-GENE is silent. (C) In Cre-expressing cells, rtTA and EGFP expression is turned on. However, in the absence of an inducer (e.g. doxycycline), rtTA cannot activate the expression of the tet-O-GENE target. Addition of doxycycline results in the formation of an active transactivator and expression of the target gene. In the present study, VEGF-A-164 was induced either ubiquitously or nervous system or podocyte specifically in the presence of doxycycline.
Figure 3
Figure 3
Tissue-specific expression of the rtTA-IRES-EGFP bicistronic transgene is Cre-dependent and the rtTA trans-activation of tet-O-VEGF-A-164 is tightly regulated by doxycycline. (A–C) Images of triple transgenic pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ E9.5 embryo and yolk sac in the absence of doxycycline: (A), Whole mount bright field microscopy; (B), GFP fluorescence microscopy; and (C), H&E histological section of the yolk sac. In the absence of doxycycline, triple transgenic embryos develop normally. Arrow in (A) shows normal yolk sac vessel with primitive hemoglobin-containing RBCs and normal blood island (BI) formation between the primitive endoderm (e) and mesoderm (m) layers of the yolk sac (C). (D–F) Triple transgenic embryo and yolk sac, the mother treated with doxycycline. (D), Whole mount bright field microscopy; (E), GFP fluorescence microscopy; and (F), H&E histological section of the yolk sac. (D) In the presence of doxycycline the triple transgenic embryos show a lethal phenotype with no proper vessel development in the yolk sac (ys) and embryo (e), no hemoglobin-containing RBC (D) and abnormal blood island (BI) structures that are filled with excessive nucleated erythroblasts (EB). (B and E) The overall EGFP signal marks the overall rtTA expression in the yolk sac and embryo of the triple transgenic embryos. (G) E10.5 triple transgenic Nestin-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ embryos develop normally in the absence of doxycycline and show nervous system specific expression of EGFP/rtTA. (H and I) E12.5 triple transgenic embryos when the mother was either non-treated (H) or treated (I) with doxycycline during the pregnancy. The VEGF-A-164 induced embryo (I) shows hemorrhages in the developing nervous system (arrows). (J) Immunostaining for EGFP shows the podocyte specificity of Podocin-Cre excision.
Figure 4
Figure 4
Liver peliosis-like phenotype and thymus degeneration are associated with the doxycycline-induced overall VEGF-A-164 expression in adult pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ mice. H&E histological analysis of livers of triple transgenic adult mice of (A–C): VEGF-A-164 non-induced (−DOX) and (D–F): VEGF-A-164 induced (+DOX) by doxycycline treatment. Without doxycyclin treatment, livers have a normal hepatic architecture with well-defined vasculature and normal hepatic venules (V) and hepatic sinusoids (HS) (C). The doxycycline-treated mutant livers show major disruptions of normal hepatic architecture with a severe dilation of hepatic sinusoids (HS) (F) and evidence of blood engorgement and pooling [arrows in (E)]. Arrow in (F) shows evidence of sinusoidal endothelial sloughing and detachment. (G–I): H&E histological analysis of VEGF-A-164 non-induced (−DOX) and (J–L): VEGF-A-164 induced (+DOX) thymus of triple transgenic adult mice. Without doxycycline treatment the thymus shows normal thymic architecture with a well-defined cortex layer (cx) that is packed with basophilic lymphocytes and a normal medullary layer (m) that contains fewer lymphocytes but an extensive epithelial framework. Doxycycline-treated mutant thymus shows massive degeneration with all three thymic lobes fitting into a single optical field (J) compared with one thymic lobe fitting into the optic field at the same magnification (G) from control mice. In addition, the mutant thymus lacks a well-defined cortical layer owing to a dramatically decreased number of basophilic thymocytes seen at higher magnifications (L) compared with (I). (A, D, G and J), 50× magnification; (B and E), 125× magnification; (H and K), 250× magnification; and (C, F, I and L), 500× magnification.
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