Mouse Line Expressing Cre Recombinase Research Articles

Smooth Muscle At1a Receptor Mediates Perivascular Fibrosis in Angiotensin II‐infused Mice

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock‐In, KIsm22α‐Cre). Male AT1a flox/flox KIsm22α‐Cre+/‐ (SMKO) and control (AT1a flox/flox KIsm22α‐Cre‐/‐) mice were infused with Ang II (1 µg/kg/min) for 2 weeks via osmotic mini‐pump. In control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart‐to‐body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart‐to‐body weight ratios were significantly increased in control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 µm vs. 56.4 µm, respectively, p<0.0001). In conclusion, AT1a receptors expressed in VSMC could mediate Ang II‐induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined. Our data also contrast with the past manuscript showing a protective effect in AT1a flox/flox S100A4‐Cre+/‐ (fibroblast silencing) but not in AT1a flox/flox sm22α‐Cre+/‐ mice with Ang II infusion.

Abstract 374: Smooth Muscle Cell-specific Deletion Of At1a Receptor Attenuates Vascular Fibrosis In Angiotensin Ii-infused Mice

Angiotensin II (Ang II) AT1 receptor has been shown to play a critical role in several cardiovascular diseases. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. Deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affect hypertensive fibrosis. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the endogenous sm22α promoter (Kock-In, KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and control (AT1a flox/flox Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks. In control mice, Ang II infusion induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly decreased in AT1a SMKO mice compared with control mice (6.04 versus 4.89, p=0.032). Cardiac hypertrophy was seen in control mice after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKO mice. Control mice showed vascular medial hypertrophy and perivascular fibrosis in coronary arteries, whereas these phenotypic changes were attenuated in SMKO mice (96.8 μm vs. 56.4 μm, p<0.0001). Control mice with Ang II infusion showed increased area of perivascular collagen III immunostaining in aorta, which was attenuated in AT1a SMKO mice. In conclusion, AT1a receptors expressed in VSMC could mediate Ang II-induced perivascular fibrosis. Our data together with the past manuscript showing a protective effect of fibroblast AT1a silencing suggest the importance of both VSMC and fibroblast AT1a in fibrosis. However, whether our data can be fully explained by the prevention of hypertension remains to be determined.

Abstract P252: Smooth Muscle At1a Receptor Mediates Perivascular Fibrosis In Angiotensin Ii-infused Mice

Angiotensin II (Ang II) signaling via AT1 receptor has been shown to play a critical role in the pathogenesis of hypertension, cardiovascular hypertrophy and fibrosis. We have demonstrated that ADAM17 expressed in vascular smooth muscle cells (VSMC) mediates EGF receptor activation and promotes cardiac hypertrophy and perivascular fibrosis induced by Ang II. It is conceivable that Ang II signaling in VSMCs specifically initiates cardiovascular remodeling, such as hypertrophy and fibrosis. In a recent study, deficiency of smooth muscle AT1a receptors results in diminished hypertension and protection from cardiac hypertrophy induced by Ang II. However, we have limited understanding whether smooth muscle AT1a receptors affects hypertensive fibrosis in vasculature. Thus, this study was designed to elucidate the roles of the AT1a receptor in VSMCs in cardiovascular remodeling including fibrosis during Ang II stimulation using VSMC AT1a receptor deficient mice. To delete the AT1a receptor from VSMCs, we crossed C57BL/6 transgenic mouse lines expressing Cre recombinase under the control of the sm22α promoter (KIsm22α-Cre). Male AT1a flox/flox KIsm22α-Cre+/- (SMKO) and Controls (AT1a flox/flox KIsm22α-Cre-/-) mice were infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump. In Control mice, Ang II infusion for 2 weeks induced cardiac hypertrophy indicated by heart-to-body weight ratio and echocardiogram. After 2 weeks of Ang II infusion, heart-to-body weight ratios were significantly increased in Control mice compared with AT1a SMKO mice (6.04 versus 4.89, respectively, p=0.032). Cardiac wall hypertrophy was seen in Controls after 2 weeks of Ang II infusion, which was attenuated in AT1a SMKOs. Control mice (n=5) showed vascular medial hypertrophy and perivascular fibrosis, whereas these phenotypic changes were attenuated in SMKO mice (n=4). In conclusion, AT1a receptors from VSMC could mediate Ang II-induced cardiovascular hypertrophy and perivascular fibrosis. Whether the data can be fully explained by the prevention of hypertension remains to be determined, the data contrast to the past manuscript showing a protective effect in AT1a flox/flox S100A4-Cre+/- mice (fibroblast silencing) with Ang II infusion.

Functional Analysis of an Unexplored Brain Region: the Claustrum

The claustrum is a thin, sheet-like neural structure located beneath the cerebral neocortex, and has reciprocal connections with nearly all neocortical areas. It has been hypothesized to play roles in higher brain functions, such as consciousness, multisensory integration, salience detection, and attentional load allocation. However, its roles in brain physiology have not been precisely elucidated, as only a few experimental studies on claustrum function exist. We established a transgenic mouse line expressing Cre recombinase specifically in a population of claustral excitatory neurons that received inputs from and sent outputs to widespread neocortical areas. The claustral neuronal firing was mostly correlated with the cortical slow-wave activity. In vitro optogenetic stimulation of the claustrum induced excitatory postsynaptic responses in most of the neocortical neurons, however, action potentials were primarily elicited in the inhibitory interneurons. In vivo optogenetic stimulation induced a synchronized Down-state featuring prolonged silencing of neural activity in all layers across widespread cortical areas, followed by Down-to-Up state transition. In contrast, genetic ablation of the claustral neurons led to an attenuation of slow-wave activity in the frontal cortex. These results indicate a crucial role of the claustum in synchronizing inhibitory interneurons across the wide cortical areas for spatiotemporal coordination of slow-wave activity.

73 - Characterization and Flaws of a New Potential β-Cell-Specific Mouse Line

Development and thorough characterization of genetic tools is necessary for efficient and productive understanding of the beta cell. Over the past few years, multiple mouse lines expressing Cre recombinase in beta cells have been developed, but all have significant caveats towards their use. Our laboratory has invested significant effort into characterizing new and existing lines to provide a model for the beta cell community. Our goal is to provide a mouse that 1) has efficient, inducible Cre expression; 2) only in beta cells and 3) limits the need for drug administration (i.e. tamoxifen and doxycycline) during critical periods of experimentation. In this study, we characterized an inducible Tet-Off system using a combination of a bacterial artificial chromosome (BAC) transgenic Ins1-itTA with the TetO-CreJaw/J line. Unfortunately, in our characterization (including assessment of: gene recombination in multiple tissues; beta cell architecture/mass; in vitro and in vivo glucose-stimulated insulin secretion (GSIS) and whole body glucose homeostasis), we discovered a significant defect in pancreatic and islet insulin content and insulin secretion in mice expressing the Ins1-itTA transgene. Moreover, we found that the rtTA or the tTA proteins themselves decreased expression of the Insulin 1, Insulin 2 and Glut2 genes in INS-1 cells, corresponding to a significant decrease in insulin content. Overall, these results considerably decreased our enthusiasm toward using the Tet-On or Tet-Off systems for future projects in beta cell, but it also emphasizes the importance of complete characterization of new mouse lines and the use of all controls for in vivo studies.

Identification of Cardiac Expression Pattern of Transient Receptor Potential Vanilloid Type 1 (TRPV1) Receptor using a Transgenic Reporter Mouse Model

Transient receptor potential vanilloid type 1 (TRPV1) channels are structurally related, non-selective cation channels that exhibit a high permeability to calcium. Sensory nerve endings expressing TRPV1 channels play a prominent role in regulating the cardiac sympathetic afferent reflex and contribute to cardiac remodeling and dysfunction in chronic heart failure. However, the precise expression of TRPV1 channels in cardiomyocytes vs. non-cardiomyocytes remains debated. Here we utilized a tdTomato-GFP reporter mouse crossed with a mouse line expressing Cre recombinase under the control of the TRPV1 promoter to map the TRPV1 expression pattern in heart. In this model, TRPV1-negative cells express tdTomato protein (red), whereas TRPV1-positive cells express GFP protein (green). As we expected, substantial GFP expression was found in many small and medium diameter dorsal root ganglia neurons in heterozygous TRPV1-Cre +/-, tdTomato flox/flox +/- male mice, suggesting that this heterozygous model is sufficient for labeling TRPV1-positive cells. Furthermore, these results showed that GFP green staining was not detectable in cardiomyocytes. Instead, we found strong GFP green staining in cardiac blood vessels—thought to be arterioles—in the heart. We also observed strong GFP signals on PGP9.5-positive cardiac nerve endings in the epicardium. In summary, this study does not support the concept that TRPV1 channels are strongly expressed in mouse cardiomyocytes. We conclude that TRPV1 channels in mouse heart are mostly expressed on non-cardiomyocyte cells including cardiac nerve endings and vessels. These data have important implications for the modulations of cardiogenic reflexes.

Targeting Morphine-Responsive Neurons: Generation of a Knock-In Mouse Line Expressing Cre Recombinase from the Mu-Opioid Receptor Gene Locus.

The mu-opioid receptor (MOR) modulates nociceptive pathways and reward processing, and mediates the strong analgesic and addictive properties of both medicinal as well as abused opioid drugs. MOR function has been extensively studied, and tools to manipulate or visualize the receptor protein are available. However, circuit mechanisms underlying MOR-mediated effects are less known, because genetic access to MOR-expressing neurons is lacking. Here we report the generation of a knock-in Oprm1-Cre mouse line, which allows targeting and manipulating MOR opioid-responsive neurons. A cDNA encoding a T2A cleavable peptide and Cre recombinase fused to enhanced green fluorescent protein (EGFP/Cre) was inserted downstream of the Oprm1 gene sequence. The resulting Oprm1-Cre line shows intact Oprm1 gene transcription. MOR and EGFP/Cre proteins are coexpressed in the same neurons, and localized in cytoplasmic and nuclear compartments, respectively. MOR signaling is unaltered, demonstrated by maintained DAMGO-induced G-protein activation, and in vivo MOR function is preserved as indicated by normal morphine-induced analgesia, hyperlocomotion, and sensitization. The Cre recombinase efficiently drives the expression of Cre-dependent reporter genes, shown by local virally mediated expression in the medial habenula and brain-wide fluorescence on breeding with tdTomato reporter mice, the latter showing a distribution patterns typical of MOR expression. Finally, we demonstrate that optogenetic activation of MOR neurons in the ventral tegmental area of Oprm1-Cre mice evokes strong avoidance behavior, as anticipated from the literature. The Oprm1-Cre line is therefore an excellent tool for both mapping and functional studies of MOR-positive neurons, and will be of broad interest for opioid, pain, and addiction research.

Interleukin-1\u03b2 signaling in fenestrated capillaries is sufficient to trigger sickness responses in mice

BackgroundThe physiological and behavioral symptoms of sickness, including fever, anorexia, behavioral depression, and weight loss can be both beneficial and detrimental. These sickness responses are triggered by pro-inflammatory cytokines acting on cells within the brain. Previous research demonstrates that the febrile response to peripheral insults depends upon prostaglandin production by vascular endothelial cells, but the mechanisms and specific cell type(s) responsible for other sickness responses remain unknown. The purpose of the present study was to identify which cells within the brain are required for sickness responses triggered by central nervous system inflammation.MethodsIntracerebroventricular (ICV) administration of 10 ng of the potent pro-inflammatory cytokine interleukin-1β (IL-1β) was used as an experimental model of central nervous system cytokine production. We examined which cells respond to IL-1β in vivo via fluorescent immunohistochemistry. Using multiple transgenic mouse lines expressing Cre recombinase under the control of cell-specific promoters, we eliminated IL-1β signaling from different populations of cells. Food consumption, body weight, movement, and temperature were recorded in adult male mice and analyzed by two-factor ANOVA to determine where IL-1β signaling is essential for sickness responses.ResultsEndothelial cells, microglia, ependymal cells, and astrocytes exhibit nuclear translocation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in response to IL-1β. Interfering with IL-1β signaling in microglia, endothelial cells within the parenchyma of the brain, or both did not affect sickness responses. Only mice that lacked IL-1β signaling in all endothelium including fenestrated capillaries lacked sickness responses.ConclusionsThese experiments show that IL-1β-induced sickness responses depend on intact IL-1β signaling in blood vessels and suggest that fenestrated capillaries act as a critical signaling relay between the immune and nervous systems.Trial registrationNot applicable.

Hypothalamic Tuberomammillary Nucleus Neurons: Electrophysiological Diversity and Essential Role in Arousal Stability.

Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons (Hdc-Cre) followed by a systematic census of the membrane properties of both HA and non-HA neurons in the ventral TMN (TMNv) region. Through unsupervised hierarchical cluster analysis, we found electrophysiological diversity both between TMNv HA and non-HA neurons, and among HA neurons. To directly determine the impact of acute cessation of HA neuron activity on sleep-wake states in awake and behaving mice, we examined the effects of optogenetic silencing of TMNv HA neurons in vivo We found that acute silencing of HA neurons during wakefulness promotes slow-wave sleep, but not rapid eye movement sleep, during a period of low sleep pressure. Together, these data suggest that the tonic firing of HA neurons is necessary for the maintenance of wakefulness, and their silencing not only impairs arousal but is sufficient to rapidly and selectively induce slow-wave sleep.SIGNIFICANCE STATEMENT The function of monoaminergic systems and circuits that regulate sleep and wakefulness is often disrupted as part of the pathophysiology of many neuropsychiatric disorders. One such circuit is the posterior hypothalamic histamine (HA) system, implicated in supporting wakefulness and higher brain function, but has been difficult to selectively manipulate owing to cellular heterogeneity in this region. Here we use a transgenic mouse to interrogate both the characteristic firing properties of HA neurons and their specific role in maintaining wakefulness. Our results demonstrate that the acute, cell type-specific silencing of HA neurons during wakefulness is sufficient to not only impair arousal but to rapidly and selectively induce slow-wave sleep. This work furthers our understanding of HA-mediated mechanisms that regulate behavioral arousal.

Metabolic Characterization of a Novel RORα Knockout Mouse Model without Ataxia.

The retinoic acid receptor-related receptor α (RORα) is a nuclear receptor that plays an important role in regulation of metabolism and the immune system. Genetic deletion of the receptor yields mice with significant cerebellar developmental issues associated with severe ataxia. Although many metabolic studies have been performed in these models, the impaired locomotor activity of these mice is known to affect their normal mobility and feeding behaviors. This creates some difficulty in interpretation of the role of RORα in models of metabolic disease where feeding and muscle function is a critical component of the pathophysiology. We generated a mouse with a floxed Rora allele that we crossed with a mouse line expressing Cre recombinase under the control of the EIIa promoter to obtain a full body deletion of Rora. This cross led to a partial deletion of the Rora locus likely due to mosaic expression of the EIIa-Cre transgene. These mice lack any signs of ataxia but display an improved metabolic profile relative to normal WT mice. The mice were resistant to diet- and age-induced metabolic syndrome and exhibited improved glucose tolerance and increased insulin sensitivity. Decreased RORα expression in the mice was also associated with reduced inflammation in models of metabolic syndrome. These data indicate that suppression of RORα activity improves metabolic function and reduces inflammation.

Phenotypically silent Cre recombination within the postnatal ventricular conduction system.

The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3’ untranslated region (3’UTR). Here we show that Cntn23’UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23’UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.

Genetic ablation of placental sinusoidal trophoblast giant cells causes fetal growth restriction and embryonic lethality

A specialized subtype of trophoblast giant cells (TGCs) line the torturous sinusoids of the murine placental labyrinth, and can be distinguished from most other TGCs by the expression of Ctsq. We generated a transgenic mouse line expressing Cre recombinase from the Ctsq promoter. Crosses with Cre-inducible tdTomato reporter mice indicated Cre activity was restricted to the sinusoidal TGCs of the labyrinth, as well as the recently characterized channel TGCs. When crossed with Cre-inducible DTA transgenic mice, ablation of sinusoidal TGCs was achieved in double transgenic embryos, resulting in fetal growth restriction by E16.5, and embryonic lethality by term.

A Transgenic Prox1-Cre-tdTomato Reporter Mouse for Lymphatic Vessel Research

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.

Functional imaging of cortical feedback projections to the olfactory bulb.

Processing of sensory information is substantially shaped by centrifugal, or feedback, projections from higher cortical areas, yet the functional properties of these projections are poorly characterized. Here, we used genetically-encoded calcium sensors (GCaMPs) to functionally image activation of centrifugal projections targeting the olfactory bulb (OB). The OB receives massive centrifugal input from cortical areas but there has been as yet no characterization of their activity in vivo. We focused on projections to the OB from the anterior olfactory nucleus (AON), a major source of cortical feedback to the OB. We expressed GCaMP selectively in AON projection neurons using a mouse line expressing Cre recombinase (Cre) in these neurons and Cre-dependent viral vectors injected into AON, allowing us to image GCaMP fluorescence signals from their axon terminals in the OB. Electrical stimulation of AON evoked large fluorescence signals that could be imaged from the dorsal OB surface in vivo. Surprisingly, odorants also evoked large signals that were transient and coupled to odorant inhalation both in the anesthetized and awake mouse, suggesting that feedback from AON to the OB is rapid and robust across different brain states. The strength of AON feedback signals increased during wakefulness, suggesting a state-dependent modulation of cortical feedback to the OB. Two-photon GCaMP imaging revealed that different odorants activated different subsets of centrifugal AON axons and could elicit both excitation and suppression in different axons, indicating a surprising richness in the representation of odor information by cortical feedback to the OB. Finally, we found that activating neuromodulatory centers such as basal forebrain drove AON inputs to the OB independent of odorant stimulation. Our results point to the AON as a multifunctional cortical area that provides ongoing feedback to the OB and also serves as a descending relay for other neuromodulatory systems.

Prostate Epithelium-Specific Deletion of the Selenocysteine tRNA Gene Trsp Leads to Early Onset Intraepithelial Neoplasia

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.

Characterization of a novel transgenic mouse line expressing Cre recombinase under the control of the Cdx2 neural specific enhancer

Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal-related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R-YFP reporter mice have indicated that the Cdx2NSE-Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double-transgenic embryos has revealed that this novel Cre-driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso-ventral axis (including pre-migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non-cranial neural crest derivatives of double-transgenic embryos. Therefore, we believe that the Cdx2NSE-Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems.

Striatum-specific expression of Cre recombinase using the Gpr88 promoter in mice

We generated a transgenic (Tg) mouse line expressing Cre recombinase under the control of the Gpr88 promoter within a bacterial artificial chromosome clone. We crossed the established Tg mice with reporter mice (CAG-CAT-Z Tg), which express Escherichia coli lacZ in response to Cre-mediated excision of the loxP-flanked chloramphenicol acetyltransferase gene, and examined the Cre activity in the Tg mouse brains by assessing β-galactosidase activity. Cre activity was specifically detected in the caudate-putamen, nucleus accumbens, and olfactory tubercle of the Gpr88-Cre Tg mouse brain. Medium spiny neurons within the caudate-putamen exhibited Cre activity. Thus, Gpr88-Cre Tg mice could be a useful tool for analyzing the function of the basal ganglia by using Cre/loxP systems.

Creating conditional knockout mice for OA research

s / Osteoarthritis and Cartilage 21 (2013) S1–S8 S7 I-22 CREATING CONDITIONAL KNOCKOUT MICE FOR OA RESEARCH P.J. Roughley. Shriners Hosp., Montreal, QC, Canada Purpose: Traditional means for creating OA in animal models has involved the use of surgery to destabilize the joint or the creation of joint inflammation, both of which result in subsequent cartilage degeneration. However, while these methods allow the progression of cartilage degeneration to be studied, they do not permit the necessity of individual molecules to be established. The use of knockout mice can potentially solve this problem. However, global knockouts are often not useful as they may have lethal consequences during development due to the involvement of multiple organs. This led to the development of conditional knockout mice, in which gene deletion is confined to cartilage. Even cartilage-specific knockout can be problematic, when the gene of interest affects chondrogenesis or growth, but this can be overcome by the use of inducible conditional knockout mice. This workshop will describe how such mice can be generated. Methods: Gene knockout is based on the ability to excise genomic regions that are essential for protein expression or function, and the Cre/LoxP system is most commonly used for this purpose. This involves the creation of a Floxed mouse line by the insertion of LoxP sites around the gene region of interest, and the subsequent breeding with a mouse line expressing Cre recombinase to excise at the LoxP sites and then recombine the excised gene. Cartilage-specificity is achieved having Cre expression under control of a cartilage specific gene promoter, such as that for type II collagen (Col2a1). For additional temporal selectivity, Cre expression is also placed under control of either doxycycline or tamoxifen administration. With this system, knockout can be achieved at any time throughout life, as long as the promoter driving Cre expression remains active. Results: Cartilage-specific knockout was initially achieved using the Col2a1 promoter to drive Cre expression, which allows gene knockout in both the articular cartilages of all diathrodial joints and the intervertebral discs of the spine. Twomodifications to this system have been developed to facilitate inducible Cre expression. In the first, Cre is expressed as a fusion protein in conjunctionwith the estrogen receptor, and is only translocated to the nucleus upon tamoxifen administration. In the second, Cre expression is placed under control of rtTA expression which in turn is driven by the Col2a1 promoter, with rtTA function being dependent upon doxycycline administration. Use of the Col2a1 promoter is, however, limited to early adult life as the expression of this promoter declines with age. This limitation has been overcome in two systems by use of the aggrecan promoter or enhancer. In one system, the Cre/estrogen receptor cassette has been place within the 30-UTR of the endogenous aggrecan gene, and in the other, the aggrecan enhancer has been used to ensure prolonged activity of the Col2a1 promoter. Conclusions: Conditional knockout mice provide a means of studying the participation of individual molecules in the progression of cartilage degeneration, and when coupled with temporal selectivity this can be performed at any stage of the disease process. The knockoutmice can be used directly or in combination with traditional surgical or biochemical intervention to induce the OA process. I-23 IMPACT OF INJURY ON JOINT STRUCTURE R.B. Frobell. Orthopedics, Clinical Sci., Lund Univ., Lund, Sweden Purpose: Knee trauma is a well-recognized and established risk factor for OA development in the longer term. The purpose of this presentation is to give an overview of trauma related structural injuries and change possibly involved the early OA disease process. Methods: Using MR imaging techniques and biochemical analysis in longitudinal trials starting in the acute phase after injury, we have monitored the structural and biochemical response of injury with the aim of identifying early changes possibly related to subsequent development of OA. Results: This presentation will give an overview of the current publications of the structural and biochemical response to knee trauma and present possible early signs of change that may be related to subsequent development of OA. Conclusions: Early changes of bone shape and cartilage thickness occur after ACL injury although the relation to later OA development remains to be determined. There also seems to be evidence in support of a biochemical alterations in the early phase of injury however, few studies are longitudinal and even fewer have correlated biochemical and structural change in this very early phase of OA. I-24 EMERGING PARADIGM: ROLES OF INFLAMMATION IN OA R. Terkeltaub y,z. yUniv. of California, San Diego, CA, USA; zVAMC, San

Lcn5 Promoter Directs the Region-Specific Expression of Cre Recombinase in Caput Epididymidis of Transgenic Mice1

Epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. To study the function of genes in the caput epididymidis using the Cre/loxP system, we generated Lcn5-Cre transgenic mice in which the expression of Cre recombinase is driven by the 1.8-kb Lcn5 promoter. A total of 11 founder mice carrying the Lcn5-Cre transgene were identified by PCR from 38 offspring, and the integration efficiency was 28.9%. However, only 1 of the 11 transgenic mouse lines were revealed with the Cre recombinase expressed specifically in caput epididymidis. Furthermore, expression of Cre mRNA was first observed on Postnatal Day 30 and continued to increase during development. Subsequently, Cre protein distribution was assessed by crossing Lcn5-Cre transgenic mice into the mT/mG reporter line. As expected, the Cre recombinase activity was only found in principal cells of the middle/distal caput epididymidis. The tissue-specific expression of Cre protein in the caput epididymidis was further confirmed using Lcn5-Cre mice crossed with a mouse strain carrying Aip1 conditional alleles (Aip1(flox/+)). In summary, a transgenic mouse line expressing Cre recombinase in principal cells of caput epididymidis was established. This transgenic mouse line can be used to generate conditional, caput epididymidis-specific knockout mouse models by crossing with mice harboring floxed (LoxP flanked) genes.

GABAergic neurons regulate lateral ventricular development via transcription factor Pax5

Postmortem studies have revealed a downregulation of the transcription factor Pax5 in GABAergic neurons in bipolar disorder, a neurodevelopmental disorder, raising the question whether Pax5 in GABAergic neurons has a role in normal brain development. In a genetic approach to study functions of Pax5 in GABAergic neurons, Pax5 was specifically deleted in GABAergic neurons from Pax5 floxed mice using a novel Gad1-Cre transgenic mouse line expressing Cre recombinase in Gad1-positive, that is, GABAergic neurons. Surprisingly, these mice developed a marked enlargement of the lateral ventricles at approximately 7 weeks of age, which was lethal within 1-2 weeks of its appearance. This hydrocephalus phenotype was observed in mice homozygous or heterozygous for the Pax5 conditional knockout, with a gene dosage-dependent penetrance. By QTL (quantitative trait loci) mapping, a 3.5 Mb segment on mouse chromosome 4 flanked by markers D4Mit237 and D4Mit214 containing approximately 92 genes including Pax5 has previously been linked to differences in lateral ventricular size. Our findings are consistent with Pax5 being a relevant gene underlying this QTL phenotype and demonstrate that Pax5 in GABAergic neurons is essential for normal ventricular development.