RESUMEN
P21-activated kinase 3 (PAK3) gene mutations are linked to several neurodevelopmental disorders, but the underlying mechanisms remain unclear. In this study, we used a tetracycline-inducible system to control the expression of a mutant PAK3 (mPAK3) protein in immediate early gene, namely cFos, positive cells to disrupt PAK signaling, specifically in cells activated by social interaction in transgenic mice. We show that the expression of mPAK3-GFP proteins was in cFos-expressing excitatory and inhibitory neurons in various brain regions, such as the cortex and hippocampus, commonly activated during learning and memory. Basal expression of mPAK3-GFP proteins in cFos-positive cells resulted in social recognition memory deficits in the three-chamber social interaction test, without affecting locomotor activity or other forms of memory. The social memory deficit was rescued by doxycycline to halt the mPAK3-GFP transgene expression. In addition, we show that the expression of mPAK3-GFP proteins in a subset of cFos-positive cells, induced by an antecedent short social interaction, termed social pairing, was sufficient to impair social recognition memory. These results indicate that normal PAK signaling in cFos-positive cells activated during social interaction is critical for social memory.
Asunto(s)
Memoria , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Interacción Social , Quinasas p21 Activadas/metabolismo , Animales , Ansiedad/fisiopatología , Conducta Animal , Doxiciclina/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Locomoción , Ratones Transgénicos , Neuronas/metabolismo , Olfato , TransgenesRESUMEN
IL-22 is an IL-10 family cytokine that is increased in asthma and atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of allergic lung inflammation and AD in vivo has yet to be elucidated. We aimed to develop mouse models of allergic diseases in the lung and skin with inducible and tissue-specific expression of IL-22, using a tetracycline (Tet)-controlled system. In this chapter, we describe a series of protocols we have developed to generate a construct that contains the TRE-Tight promoter and mouse IL-22 cDNA based on this system. Furthermore, we describe how to generate TRE-Tight-IL-22 mice through pronuclear microinjection. In our approach, two Tet-on (CC10-rtTA or SPC-rtTA) and a Tet-off (K5-tTA) transgenic mouse lines are selected to crossbreed with TRE-Tight-IL-22 mice to generate inducible tissue-specific transgenic lines. The transgenic strains, CC10-rtTA/TRE-Tight-IL-22 (CC10-rtTA-IL-22) or SPC-rtTA/TRE-Tight-IL-22 (SPC-rtTA-IL-22) mice, do not produce detectable levels of IL-22 in their bronchoalveolar lavage (BAL) samples in the absence of doxycycline (Dox). However, oral Dox treatment of these mice induces IL-22 expression in the BAL, and the airway and lung epithelial cells. For K5-tTA/TRE-Tight-IL-22 (K5-tTA-IL-22) mice, to avoid potential IL-22 toxicity to mouse embryos, Dox is given starting at the time of breeding to suppress tTA and to keep the IL-22 transgene off until the K5-tTA-IL-22 mice are 6 weeks old. Experiments are then initiated by withdrawing Dox from the drinking water. In all cases, IL-22 protein can be detected by immunohistochemistry in the skin of Tg(+) animals, but not in the skin of Tg(-) animals. Utilizing transgenic technology based on the Tetracycline-controlled system, we have established inducible transgenic mouse models in which cytokine IL-22 can be expressed specifically in the lung or skin. These models are valuable for studies in vivo in a broad range of diseases involving IL-22 and will provide a new platform for research and for seeking novel therapeutics in the fields of inflammation, asthma, and allergic dermatitis.
Asunto(s)
Transferencia de Embrión/métodos , Embrión de Mamíferos/efectos de los fármacos , Regulación de la Expresión Génica , Interleucinas/genética , Pulmón/inmunología , Piel/inmunología , Transfección/métodos , Células A549 , Animales , Doxiciclina/farmacología , Embrión de Mamíferos/citología , Embrión de Mamíferos/inmunología , Femenino , Humanos , Interleucinas/agonistas , Interleucinas/inmunología , Lípidos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microinyecciones/métodos , Plásmidos/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Transcripción Genética , Transgenes , Cigoto/citología , Cigoto/crecimiento & desarrollo , Cigoto/metabolismo , Interleucina-22RESUMEN
Social interactions are essential to our mental health, and a deficit in social interactions is a hallmark characteristic of numerous brain disorders. Various subregions within the medial temporal lobe have been implicated in social memory, but the underlying mechanisms that tune these neural circuits remain unclear. Here, we demonstrate that optical activation of excitatory entorhinal cortical perforant projections to the dentate gyrus (EC-DG) is necessary and sufficient for social memory retrieval. We further show that inducible disruption of p21-activated kinase (PAK) signaling, a key pathway important for cytoskeletal reorganization, in the EC-DG circuit leads to impairments in synaptic function and social recognition memory, and, importantly, optogenetic activation of the EC-DG terminals reverses the social memory deficits in the transgenic mice. These results provide compelling evidence that activation of the EC-DG pathway underlies social recognition memory recall and that PAK signaling may play a critical role in modulating this process.
Asunto(s)
Giro Dentado/fisiología , Corteza Entorrinal/fisiología , Recuerdo Mental/fisiología , Conducta Social , Animales , Giro Dentado/efectos de los fármacos , Corteza Entorrinal/efectos de los fármacos , Recuerdo Mental/efectos de los fármacos , Ratones Transgénicos , Optogenética , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Reconocimiento en Psicología/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/metabolismoRESUMEN
Constitutive gene expression is not always the appropriate expression system because the unphysiological levels of expressed protein could be detrimental in studies examining biological roles of proteins, or continued expression may be unnecessary after therapeutic effects have been achieved in gene therapy . We have utilized pharmacologically regulated gene expression systems to achieve fine control of gene expression levels which facilitate research in basic biology and translates to use in experimental gene therapy studies. In this chapter, we outline the application of a tightly controlled tetracycline responsive gene expression system.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Tetraciclina/farmacología , Transfección/métodos , Western Blotting/métodos , Línea Celular , Vectores Genéticos/genética , Humanos , Regiones Promotoras Genéticas/efectos de los fármacosRESUMEN
Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405 nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1 µg/mL and removing doxycycline from the media 60 min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation.