Generation of a tightly regulated doxycycline-inducible model for studying mouse intestinal biology

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.     

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.     

Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice

CD4+ T cells with their growing list of effector and regulatory subpopulations have vital functions within the immunohematopoietic system. We report here on the first mouse lines that allow temporally and quantitatively controlled expression of transgenes specifically in CD4+ thymocytes and T cells. These were constructed using the Tet-on system. The rtTA2(S)-M2 version of the reverse tetracycline-dependent transactivator was placed under control of all known CD4 regulatory elements. Reporter transgene expression in mice expressing these constructs is highly specific for CD4+ cells, is strictly dependent on the tetracycline derivative doxycycline, and can be regulated by up to five logs depending on the doxycycline concentration. Moreover, we demonstrate that these mice can be used for noninvasive in vivo imaging of a coexpressed luciferase reporter. These new mouse lines should be highly valuable for studying and manipulating numerous aspects of CD4+ T cell development, biology, and function.     

Inducible cardiomyocyte‐specific gene disruption directed by the rat Tnnt2 promoter in the mouse

We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by coinjection of two transgenes, a “reverse” tetracycline‐controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline‐responsive promoter (TetO). Here, Tnnt2‐rtTA activated TetO‐Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte‐specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2‐rtTA;TetO‐Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods. genesis 48:63–72, 2010. © 2009 Wiley‐Liss, Inc.     

Sox10-rtTA mouse line for tetracycline-inducible expression of transgenes in neural crest cells and oligodendrocytes

Using gene targeting, we inserted a high-affinity variant of the reverse tetracycline controlled transactivator (rtTA) into the genomic Sox10 locus. This rtTA transgene faithfully recapitulated Sox10 expression in the emerging neural crest, several of its derivatives, and in oligodendrocytes. It was furthermore able to induce expression of a tetracycline inducible transgenic reporter gene in a doxycycline-dependent manner. Induction was fast, with substantial reporter gene expression visible 6 h after the onset of doxycycline treatment. Shut-off, in contrast, exhibited delayed kinetics, which probably correlated with doxycycline clearance rates. This mouse provides a useful tool for generating tetracycline-controlled gene expression in neural crest and oligodendrocytes.     

Inducible and neuron-specific gene expression in the adult mouse brain with the rtTA2S-M2 system

To achieve inducible and reversible gene expression in the adult mouse brain, we exploited an improved version of the tetracycline-controlled transactivator-based system (rtTA2(S)-M2, rtTA2 hereafter) and combined it with the forebrain-specific CaMKIIalpha promoter. Several independent lines of transgenic mice carrying the CaMKIIalpha promoter-rtTA2 gene were generated and examined for anatomical profile, doxycycline (dox)-dependence, time course, and reversibility of gene expression using several lacZ reporter lines. In two independent rtTA2-expressing lines, dox-treatment in the diet induced lacZ reporter expression in neurons of several forebrain structures including cortex, striatum, hippocampus, amygdala, and olfactory bulb. Gene expression was dose-dependent and was fully reversible. Further, a similar pattern of expression was obtained in three independent reporter lines, indicating the consistency of gene expression. Transgene expression could also be activated in the developing brain (P0) by dox-treatment of gestating females. These new rtTA2-expressing mice allowing inducible and reversible gene expression in the adult or developing forebrain represent useful models for future genetic studies of brain functions.     

Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart

Tetracycline regulated gene expression in transgenic animals is potentially a very powerful technique (Furth et al., 1994; Gossen & Bujard 1992). We have utilized this system in an attempt to overcome the perinatal lethality resulting from constitutive transgenic expression in the heart (Valencik & McDonald, Am J Physiol Heart Circ Physiol 280: H361–H367). We found that compound hemizygous animals created by mating selected reverse tetracycline transactivator (rtTA) and transresponder (TR) lines display tightly regulated TR expression in the heart. However, we identified two fundamental problems. First, codon usage bias appeared to severely limit the expression of the rtTA driven by the cardiac -myosin heavy chain promoter. Second, co-injection of rtTA and TR transgenes led to compound hemizygous animals that exhibited unregulated TR gene expression. Codon optimization of the rtTA construct leads to marked improvement (increasing the average induction from 20-fold to 832-fold) in cardiac myocyte expression. The resulting opt-rtTA lines can be bred to homozygosity, facilitating rapid screening of F0 TR animals for doxycycline regulated transgene expression.     

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet‐on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET‐ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.     

Identification of quiescent, stem-like cells in the distal female reproductive tract

In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature Műllerian epithelial cells. Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct. Future lineage-tracing studies will elucidate the role played by these cells in homeostasis, tissue injury and cancer of the female reproductive tract in the mouse and eventually in man.     

The reverse tetracycline-controlled transactivator rtTA2s-S2 is toxic in mouse embryonic stem cells

The efficient and reversible control of transgene expression is a powerful tool for the correct manipulation of embryonic stem cells in both cell therapy and transgenesis. The aim of this work was to investigate the possibilities of recently developed reverse tetracycline-controlled transactivator rtTA2s-S2. We show that the rtTA2s-S2 is useful for transient inducible expression of genes in embryonic stem cells. However, we found that it was not possible to establish mouse embryonic stem cell lines stably expressing this transactivator. Using the viral IRES sequence which couples the expression of rtTA2s-S2 and neomycin phosphotransferase, we found that embryonic stem cells expressing rtTA2s-S2 are not capable of growing in the presence of G418. Our results indicate that this transactivator is toxic to ES cells and raise the need for the development of other strategies for stable and inducible expression of genes in ES cells.     

Inducible gene modification in the gastric epithelium of Tff1‐CreERT2, Tff2‐rtTA,Tff3‐luc mice

Temporal and spatial regulation of genes mediated by tissue‐specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium‐specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain confers tamoxifen‐inducible irreversible somatic recombination and allows simultaneous doxycycline‐dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1‐CreERT2 and Tff2‐rtTA transgene activity in a partially overlapping subset of long‐term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626–635, 2016. © 2016 Wiley Periodicals, Inc.