Inducible,tightly regulated and non-leaky neuronal gene expression in mice

The Tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgene expression in mice with over 500 lines created to date. Although this system has been optimized over the years, it still has limitations such as residual transgene expression when turned off, referred to as leakiness. Here, we present a series of new Tet-OFF transgenic mice based on the second generation tetracycline-responsive transactivator system. The tTA-Advanced (tTA2^S) is expressed under control of the neuron-specific Thy1.2 promoter (Thy-OFF), to regulate expression in the mouse brain. In addition, we generated a lacZ reporter line, utilizing the P _tight Tet-responsive promoter (P _tight–lacZ), to test our system. Two Thy-OFF transgenic lines displaying two distinct patterns of expression were selected. Oral doxycycline treatment of Thy-OFF/P _tight–lacZ mice demonstrated tight transgene regulation with no leak expression. These new Thy-OFF mice are valuable for studies in a broad range of neurodegenerative diseases such as Alzheimer’s disease and related forms of dementia, where control of transgene expression is critical to understanding mechanisms underlying the disease. Furthermore, P _tight–lacZ reporter mice may be widely applicable.     

Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1beta, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.     

Transgenic mice carrying a tetracycline-inducible, truncated transforming growth factor beta receptor (TbetaRII)

The transforming growth factor-betas (TGFbetas) have multiple roles, making genetic analysis of their functions difficult. We therefore developed transgenic mouse lines to disrupt TGFbeta signaling using a mechanism that is inducible, reversible, and cell-type specific. The transgenic mouse lines carry an EGFP-pBi-DeltaTbetaRII construct (PTR). The DeltaTbetaRII element codes for a dominant-negative receptor that is known to disrupt TGFbeta signaling. The DeltaTbetaRII has a c-myc tag. The transgene was silent in the PTR mice, with expression of both EGFP and DeltaTbetaRII occurring when the PTR mice were crossed with mice that express the tetracycline transactivator (CMV-tTA). The expression of EGFP was repressed by the addition of doxycycline to the drinking water of the PTRxCMV-tTA mice. The PTR mice were then crossed with neuron-specific-tTA mice. Expression of the DeltaTbetaRII transgene in these mice led to an upregulation of native TGFbeta receptor expression, suggesting that neurons can modulate their responsiveness to TGFbetas.     

Tetracycline inducible gene manipulation in serotonergic neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.     

Transgenic expression of CTLA-4 controls lymphoproliferation in IL-2-deficient mice

IL-2-deficient mice develop a lymphoproliferative and autoimmune disease characterized by autoimmune hemolytic anemia (AHA) and inflammatory bowel disease. We have previously reported that IL-2 is necessary for optimal up-regulation of CTLA-4, an inducible negative regulator of T cell activation. In this study, we have tested the hypothesis that reduced expression of CTLA-4 in IL-2-deficient T cells contributes to the pathogenesis of disease in IL-2-deficient mice. Expression of CTLA-4 as a transgene completely prevented lymphoaccumulation and AHA in IL-2-deficient mice. The normalization of T cell numbers was due to inhibition of expansion of conventional CD4+CD25- T cells rather than to rescue of the numbers or function of CD4+CD25+ regulatory T cells, suggesting that CTLA-4 expression on conventional T cells plays a role in maintaining normal T cell homeostasis. In addition, the inhibitory effect of the CTLA-4 transgene on T cell expansion was at least in part independent of CD28 expression. Our results suggest that deficient CTLA-4 expression on conventional T cells contributes to the pathophysiology of the lymphoproliferative disease and AHA in IL-2-deficient mice. Thus, restoring CTLA-4 expression in T cells may be an attractive strategy to control clinical autoimmune diseases in which CTLA-4 expression is reduced.     

Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island

Background  

A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice.     

CaMKII activation in the entorhinal cortex disrupts previously encoded spatial memory

To investigate the role of the entorhinal cortex in memory at a molecular level, we developed transgenic mice in which transgene expression was inducible and limited to the superficial layers of the medial entorhinal cortex, pre- and parasubiculum. We found that expression of a constitutively active mutant form of CaMKII in these structures disrupted spatial memory formation. Immediate post-training activation of the transgene disrupted previously established memory while transgene activation 3 weeks following the training was ineffective. These results demonstrate that, similar to the hippocampus, the entorhinal cortex plays a time-limited role in spatial memory formation but is not a final cortical repository of long-term memory. Moreover, these results suggest that the indiscriminate activation of CaMKII is able to disrupt preexisting memories, possibly by altering the pattern of synaptic weight changes that are thought to form the basis of the memory trace.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.     

Regulation of Endothelial-Specific Transgene Expression by the LacI Repressor Protein In Vivo

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI^R, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI^R protein.     

Efficient inducible Pan-neuronal cre-mediated recombination in SLICK-H transgenic mice

Large-scale functional genomics in mice is becoming feasible through projects to develop conditional knockout alleles for every gene. Inducible neuron-specific gene knockout in such mice will permit the analysis of neuronal phenotypes while circumventing developmental defects or embryonic lethality. Here we describe a transgenic line, termed SLICK-H, that facilitates widespread inducible conditional genetic manipulation within most populations of projection neurons. In SLICK-H mice, the Thy1 promoter drives robust and relatively uniform expression of a drug-inducible form of cre recombinase throughout the peripheral and central nervous system. This permits efficient induction of cre-mediated genetic manipulation upon tamoxifen administration in adult mice. Importantly, cre activity in the absence of tamoxifen is minimal, permitting tight control of recombination. In the present study, we catalog in detail the transgene expression patterns and recombination efficiencies in SLICK-H mice. Our results highlight the utility of SLICK-H mice for functional genomics in the nervous system.     

Functional expression of a heterologous major histocompatibility complex class I gene in transgenic mice.

The regulated expression of major histocompatibility complex class I antigens is essential for assuring proper cellular immune responses. To study H-2 class I gene regulation, we have transferred a foreign class I gene to inbred mice and have previously shown that the heterologous class I gene was expressed in a tissue-dependent manner. In this report, we demonstrate that these mice expressed the transgenic class I molecule on the cell surface without any alteration in the level of endogenous H-2 class I antigens. Skin grafts from transgenic mice were rapidly rejected by mice of the background strain, indicating that the transgenic antigen was expressed in an immunologically functional form. As with endogenous H-2 class I genes, the class I transgene was inducible by interferon treatment and suppressible by human adenovirus 12 transformation. Linkage analysis indicated that the transgene was not closely linked to endogenous class I loci, suggesting that trans-regulation of class I genes can occur for class I genes located outside the major histocompatibility complex.     

A new mouse model for temporal‐ and tissue‐specific control of extracellular superoxide dismutase

The extracellular isoform of superoxide dismutase (EC‐SOD, Sod3) plays a protective role against various diseases and injuries mediated by oxidative stress. To investigate the pathophysiological roles of EC‐SOD, we generated tetracycline‐inducible Sod3 transgenic mice and directed the tissue‐specific expression of transgenes by crossing Sod3 transgenic mice with tissue‐specific transactivator transgenics. Double transgenic mice with liver‐specific expression of Sod3 showed increased EC‐SOD levels predominantly in the plasma as the circulating form, whereas double transgenic mice with neuronal‐specific expression expressed higher levels of EC‐SOD in hippocampus and cortex with intact EC‐SOD as the dominant form. EC‐SOD protein levels also correlated well with increased SOD activities in double transgenic mice. In addition to enabling tissue‐specific expression, the transgene expression can be quickly turned on and off by doxycycline supplementation in the mouse chow. This mouse model, thus, provides the flexibility for on–off control of transgene expression in multiple target tissues. genesis 47:142–154, 2009. © 2009 Wiley‐Liss, Inc.     

Generation and evaluation of an IPTG-regulated version of Vav-gene promoter for mouse transgenesis

Different bacteria-derived systems for regulatable gene expression have been developed for the use in mammalian cells and some were also successfully adopted for in vivo use in vertebrate model organisms. However, certain limitations apply to most of these systems, including leakiness of transgene expression, inefficient transgene silencing or activation, as well as limited tissue accessibility of transgene-inducers or their unfavourable pharmacokinetics. In this study, we evaluated the suitability of the lac-operon/lac-repressor (lacO/lacI) system for the regulation of the well-established Vav-gene promoter that allows inducible transgene expression in different haematopoietic lineages in mice. Using the fluorescence marker protein Venus as a reporter, we observed that the lacO/lacI system could be amended to modulate transgene-expression in haematopoietic cells. However, reporter expression was not uniform and the lacO elements introduced into the Vav-gene promoter only conferred limited repression and reversion of lacI-mediated gene silencing after administration of IPTG. Although further optimization of the system is required, the lacO-modified version of the Vav-gene promoter may be adopted as a tool where low basal gene-expression and limited transient induction of protein expression are desired, e.g. for the activation of oncogenes or transgenes that act in a dominant-negative manner.