Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

Zhu, Peixin; Aller, M. Isabel; Baron, Udo; Cambridge, Sidney B.; Bausen, Melanie; Herb, Jan T.; Sawiński, J; Çetin, Ali; Osten, Pavel; Nelson, Mark L.; Kügler, Sebastian; Seeburg, Peter H.; Sprengel, Rolf; Hasan, Mazahir T.

doi:10.1371/journal.pone.0000533

Cited by 84 publications

(114 citation statements)

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“…Litters were fostered to Dox-naïve dams (Fig. 1C), thus activating tTA, which was transgenically expressed in principal neurons of the forebrain via the α-calcium/calmodulin-dependent protein kinase II (α−CamKII) promoter (16)(17)(18). In absence of Dox, tTA induced Cre expression and, subsequently, Npy1r gene inactivation in juvenile Npy1r 2lox /Tg αCamKII-tTA/LC1 mice (Npy1r rfb mice; rfb, reduced forebrain expression).…”

Section: Resultsmentioning

confidence: 99%

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

Bertocchi

Oberto

Longo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Neuropeptide Y (NPY) plays an important role in stress, anxiety, obesity, and energy homeostasis via activation of NPY-Y1 receptors (Y1Rs) in the brain. However, global knockout of the Npy1r gene has low or no impact on anxiety and body weight. To uncover the role of limbic Y1Rs, we generated conditional knockout mice in which the inactivation of the Npy1r gene was restricted to excitatory neurons of the forebrain, starting from juvenile stages (Npy1r rfb ). Npy1r rfb mice exhibited increased anxiety and reduced body weight, less adipose tissue, and lower serum leptin levels. Npy1r rfb mutants also had a hyperactive hypothalamic-pituitaryadrenocortical axis, as indicated by higher peripheral corticosterone and higher density of NPY immunoreactive fibers and corticotropin releasing hormone immunoreactive cell bodies in the paraventricular hypothalamic nucleus. Importantly, through fostering experiments, we determined that differences in phenotype between Npy1r rfb and Npy1r 2lox mice became apparent when both genotypes were raised by FVB/J but not by C57BL/6J dams, suggesting that limbic Y1Rs are key targets of maternal care-induced programming of anxiety and energy homeostasis. where it is involved in the regulation of anxiety, stress reactions, energy balance, circadian rhythms, and cognition (1-3). Clinical studies suggest that NPY plays an important role in the response to stress and in psychiatric disorders (4). In humans, NPY haploinsufficiency is correlated with characteristic brain responses to emotional and stress challenges and with trait anxiety (5). Intracerebroventricular injection of NPY reduces both anxiety-and stress-related behavior in several animal models, an effect that is primarily mediated by Y1 receptors (Y1Rs) expressed in amygdala, hippocampus, and locus coeruleus (6-9). The implications of a role of endogenous NPY in acting via Y1R to control emotionality, mood, and stress reactions have been probed with Y1R-selective antagonists and antisense oligonucleotides (1). NPY exerts its anxiolytic-like effect in the brain via interactions with the hypothalamic-pituitaryadrenocortical (HPA) axis and corticosteroids. Indeed, a functional antagonism between NPY and corticotropin releasing hormone (CRH) has been demonstrated in various CNS nuclei along the stress/anxiety circuits (10).In addition to its crucial role in emotional behavior, NPY potently stimulates feeding, reduces energy expenditure, and induces obesity via the activation of Y1R expressed in the hypothalamus (1). However, global Npy1r gene knockout mice showed only minor deficiencies in energy homeostasis, feeding, and anxiety (11)(12)(13)(14).To study the function of Y1R expressed in the limbic system and to exclude effects induced by the Npy1r gene inactivation in early development, we restricted the ablation of Y1R to excitatory neurons in the postnatal forebrain of mice by using the Cre-loxP system (Fig. 1A). In addition, because early postnatal environment can modulate NPY levels (15), gene-targeted pups were reared by two diffe...

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Section: Resultsmentioning

confidence: 99%

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

Bertocchi

Oberto

Longo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The kinetics of the system (days) may be also too slow for some developmental problems where transient expression of developmental genes are critical, although it should be noted that it could still work well in carefully thought-out developmental studies (as in [24]). In addition, it has been reported that rtTA often can not induce sufficient gene expression in the brain, at least partially due to developmental inactivation of the TRE promoter in neurons [36]. It appears that the tTA (Tet-off) system is better suited for the use in brain [14], as substantial b-gal induction was observed in brain with tTA ( Figure 4F).…”

Section: Discussionmentioning

confidence: 94%

An Inducible and Reversible Mouse Genetic Rescue System

Santos¹

2011

Genetic Engineering

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Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and costeffective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

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“…1A). Virus with capsid proteins of the serotype 1 and 2 was prepared as described previously (Zhu et al, 2007). The culture was infected with the virus, rAAV-P hSYN -ChR2-YFP, 1 to 2 days after plating.1 We observed YFP expression in the culture at 7, 14, and 21 days in vitro (DIV).…”

Section: Results Raav Mediated Delivery Of Chr2-yfp Into Neuronsmentioning

confidence: 99%

“…To remove salt and further concentrate the virus, the idoxanol gradient fraction was washed 3X with PBS and concentrated to a volume of 200-300 μl using Amicon filters (Amersham). Infectious virus titers were determined in primary neuron cultures as described previously (Zhu et al, 2007).…”

Section: Preparation Of Raavmentioning

confidence: 99%

“…Second, long-term expression, from months to years, is achievable. Due to high rate of infectivity, rAAV can be used to introduce multiple genes into the same neurons in pre-selected brain regions (Shevtsova et al, 2005) without epigenetic silencing (Zhu et al, 2007). This broadens the experimental possibilities so that other genes whose products act as biosensors for different signaling systems, such as for calcium (Miyawaki, 2003;Palmer and Tsien, 2006;Wallace et al, 2008) and neurotransmitter release (Miesenbock et al, 1998), could also be introduced into the same neuron using rAAV as the delivery method.…”

Section: Introductionmentioning

confidence: 99%

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Laser-evoked synaptic transmission in cultured hippocampal neurons expressing channelrhodopsin-2 delivered by adeno-associated virus

Wang¹,

Hasan²,

Seung³

2009

Journal of Neuroscience Methods

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We present a method for studying synaptic transmission in mass cultures of dissociated hippocampal neurons based on patch clamp recording combined with laser stimulation of neurons expressing Channelrhodopsin-2 (ChR2). Our goal was to use the high spatial resolution of laser illumination to come as close as possible to the ideal of identifying monosynaptically coupled pairs of neurons, which is conventionally done using microisland rather than mass cultures. Using recombinant adenoassociated virus (rAAV) to deliver the ChR2 gene, we focused on the time period between 14 and 20 days in vitro, during which expression levels are high, and spontaneous bursting activity has not yet started. Stimulation by wide-field illumination is sufficient to make the majority of ChR2-expressing neurons spike. Stimulation with a laser spot at least 10 μm in diameter also produces action potentials, but in a reduced fraction of neurons. We studied synaptic transmission by voltageclamping a neuron with low expression of ChR2 and scanning a 40 μm laser spot at surrounding locations. Responses were observed to stimulation at a subset of locations in the culture, indicating spatial localization of stimulation. Pharmacological means were used to identify responses that were synaptic. Many responses were of smaller amplitude than those typically found in microisland cultures. We were unable to find an entirely reliable criterion for distinguishing between monosynaptic and polysynaptic responses. However, we propose that postsynaptic currents with Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript small amplitudes, simple shapes, and latencies not much greater than 8 msec are reasonable candidates for monosynaptic interactions.

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Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

Cited by 84 publications

References 48 publications

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

An Inducible and Reversible Mouse Genetic Rescue System

Laser-evoked synaptic transmission in cultured hippocampal neurons expressing channelrhodopsin-2 delivered by adeno-associated virus

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