eagle-i University of PennsylvaniaUniversity of Pennsylvania
See it in Search

Steven Thomas Laboratory

Summary:

Broadly, the lab studies the development and physiology of the mammalian brain. One goal is to define the systems that contribute to specific behaviors, and to understand the mechanisms that underlie these behaviors. Such knowledge may ultimately permit the prevention and treatment of mental illness. Gene-targeting allows the analysis of specific genetic alterations in the context of the whole organism. The ability to add, delete or modify genes is particularly useful in the analysis of complex organ systems such as the brain, where half of all genes are thought to be uniquely expressed.

The lab focuses on the adrenergic nervous system in which norepinephrine (NE) and epinephrine are the classic neurotransmitters. By genetically eliminating the biosynthetic enzyme for NE, dopamine beta-hydroxylase (DBH), mutant mice (Dbh-/-) that completely lack NE and epinephrine were created. These mice are conditional mutants in that NE can be restored to the adrenergic terminals by supplying a synthetic amino acid precursor of NE, L-DOPS. The lab is pursuing several fundamental observations that resulted from the creation of these mutant mice. These include the roles of NE in learning and memory, as well as the neuronal physiology and signaling that underlie these effects. They also include the role of NE in the effects of stress. For each of these, potentially important interactions with other transmitters and hormones is also being explored. Finally, Dr. Thomas is pursuing several novel genetic approaches for producing complementary models to the Dbh-/- mice toward a more complete understanding of CNS adrenergic function.

Affiliations:

People:

Resources:

Organisms and Viruses

  • Adrb1 loxP/loxP ( Mus musculus )

    Gene coding for β-adrenergic receptor 1 is floxed.

  • Adrb1-/- ( Mus musculus )

    Adrb1-/- KO mice were generated by mating either heterozygotes or homozygotes on a hybrid 129/Sv × C57BL/6 background, and the genotype was determined by PCR. These mice exhibit targeted disruption of the gene for the β1 receptor.

    "Mice that are homozygous null for the Adrb1 and Adrb2 genes are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Stimulation of beta adrenergic receptor function in these mice by agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity and metabolic rate. A severely attenuated chronotropic and hypotensive response is observed after administration of the non-selective beta adrenergic receptor agonist isoproterenol. An abnormal response to epinephrine is also seen, with bradycardia and a monophasic hypertensive blood pressure change being observed rather than the tachycardia and biphasic hypertensive/ hypotensive response seen in wildtype mice. When exercised, heart rates in null mice are lower than that of wild type mice. No difference is noted in the resting heart rate."

    "We thank Brian Kobilka (Stanford University, CA) for providing stock for the β receptor KO mouse lines."

  • Adrb2-/- ( Mus musculus )

    β2 KO mice have targeted disruption of the gene for β2-adrenergic receptor and were generated by mating either heterozygotes or homozygotes.

    "Mice that are homozygous null for the Adrb1 and Adrb2 genes are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Stimulation of beta adrenergic receptor function in these mice by agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity and metabolic rate. A severely attenuated chronotropic and hypotensive response is observed after administration of the non-selective beta adrenergic receptor agonist isoproterenol. An abnormal response to epinephrine is also seen, with bradycardia and a monophasic hypertensive blood pressure change being observed rather than the tachycardia and biphasic hypertensive/ hypotensive response seen in wildtype mice. When exercised, heart rates in null mice are lower than that of wild type mice. No difference is noted in the resting heart rate."

    "We thank Brian Kobilka (Stanford University, CA) for providing stock for the β receptor KO mouse lines."

  • Adrb3-/- ( Mus musculus )

    β3 KO mice have targeted disruption of the gene for β3-adrenergic receptor were "generated by mating either heterozygotes or homozygotes, and genotype was determined by PCR." The targeted disruption was generated in the following manner: "A 306bp genomic fragment containing the sequences encoding the third through the fifth transmembrane domains was replaced with a neomycin selection cassette."

    "We thank [...] B. Lowell for providing stock for the β receptor KO mouse lines."

  • beta1/beta2 double KO ( Mus musculus )

    These mice have targeted disruption of the gene for β1- and β2-adrenergic receptors and were generated by crossing β1 KO mice with β2 KO mice.

  • beta1/beta2/beta3 triple KO ( Mus musculus )

    These mice have targeted disruption of the gene for β1-, β2-, and β3-adrenergic receptors.

    "We thank [...] B. Kobilka and B. Lowell for providing stock for the β receptor KO mouse lines."

  • beta1/beta3 double KO ( Mus musculus )

    These mice have targeted disruption of the gene for β1- and β3-adrenergic receptors and were generated by crossing β1 KO mice with β3 KO mice.

  • CaMKII-Cre Tg ( Mus musculus )

    "Mice homozygous for the transgenic insert are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. These transgenic mice express the Cre recombinase under the control of the mouse calcium/calmodulin-dependent protein kinase II alpha promoter. Cre recombinase expression is detected in the forebrain, specifically to the CA1 pyramidal cell layer in the hippocampus. When crossed with a strain containing loxP site flanked sequence of interest, Cre-mediated recombination occurs in the pyramidal cell layer."

  • Chrm1−/− ( Mus musculus )

    "Homozygotes are viable and fertile with complete absence of the wildtype allele mRNA in forebrain tissues. Mice homozygous for this M1 muscarinic acetylcholine receptor deficiency have elevated dopaminergic transmission in the striatum, significantly increased locomotor activity, increased response to the stimulatory effects of amphetamine. These mutant mice may be useful in neurological studies, including the regulation of dopaminergic transmission by the M1 receptor and M1 dysfunction in psychiatric disorders."

  • Dat-Cre KI ( Mus musculus )

    "Mice homozygous for this dopamine transporter IRES-cre (DATIREScre) mutant allele are viable and fertile. Cre recombinase activity is observed as early as embryonic day 15, and co-localizes with endogenous gene expression in adult dopaminergic cell groups (substantia nigra (SN) and ventral tegmental area (VTA), as well as in the retrorubral field). Lesser Cre recombinase activity occurs in adult olfactory bulb glomeruli, mimicking the known lower Slc6a3 (or DAT) expression in this tissue. Although the pattern and intensity of DAT immunostaining in the SN, VTA and striatum do not differ between wild-type and mutant mice, striatum DAT protein levels are moderately reduced (17%) in heterozygotes and significantly reduced (47%) in homozygotes. This diminution in homozygous striatum is associated with significantly increased neuropeptide PDyn (but not D1, D2, or PPE) mRNA levels compared to wild-type, while such an increase is not observed in heterozygotes. When bred with mice containing a loxP-flanked sequence of interest, Cre-mediated recombination will result in deletion of the flanked genome in dopaminergic neurons. As such, these mutant mice may be useful in neurobiological studies to facilitate the analysis of gene function in dopaminergic neurons, such as drug addiction or Parkinson's disease.

    In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. It should be noted that the phenotype could vary from that originally described. The strain description will be modified as published results become available."

  • Dat-CreERT2 KI ( Mus musculus )

  • Dat-rtTA2S-M2 KI ( Mus musculus )

  • Dat-tTA2 KI ( Mus musculus )

  • Dbh-/- ( Mus musculus )

    This mouse line was generated by mating heterozygotes, and underwent targeted disruption of the dopamine β-hydroxylase gene. This resulted in complete NE/E deficiency for this mouse line, as well as retrieval deficits in spatial navigation and contextual fear.

  • Dbh-Cre KI ( Mus musculus )

  • Dbh-CreERT2 KI ( Mus musculus )

  • Dbh-rtTA2S-M2 KI ( Mus musculus )

  • Dbh-tTA2 KI ( Mus musculus )

  • Drd1-/- ( Mus musculus )

    This line was first described by Xu et al. in "Dopamine D1 receptor mutant mice are deficient in striatal expression of dynorphin and in dopamine-mediated behavioral responses" (PMID: 7954836).

    D1 KO mice have targeted disruption of the gene for dopamine receptor D1A and were "generated by mating either heterozygotes or homozygotes, and genotype was determined by PCR."

    "We thank [...] M. Xu (University of Chicago) [...] for providing stock for the [...] DA receptor KO mouse lines."

  • Drd5-/- ( Mus musculus )

    D5DR KO mice have targeted disruption of the gene for dopamine receptor D5 and were "generated by mating either heterozygotes or homozygotes, and genotype was determined by PCR."

    "A neo was inserted in reverse orientation to disrupt the reading frame within the coding region. A stop codon was engineered into the proximal neo linker to prematurely truncate the transcript subsequent to Gly-190 in the second extracellular loop. RT-PCR verified presence of the recombinant transcript in mutant brains. Immunohistochemistry using peptides from the region downstream of the stop codon showed that mutants did not produce the corresponding protein."

    "We thank [...] D. Sibley (National Institutes of Health) for providing stock for the [...] DA receptor KO mouse lines."

  • Grm5−/− ( Mus musculus )

    "Mice homozygous for the targeted mutation are viable and fertile. Long-term potentiation (LTP) in homozygous mutant mice is significantly reduced in the NMDA receptor (NMDAR)-dependent pathways such as the CA1 region and dentate gyrus of the hippocampus, whereas LTP remains intact in the mossy fiber synapses on the CA3 region, an NMDAR-independent pathway. These mutant mice also show impairments in the acquisition and use of spatial information in both the Morris water maze and contextual information in the fear-conditioning test."

  • Hdc−/− ( Mus musculus )

    "Heterozygous HDC-Cre mice are viable and fertile, expressing an internal ribosome entry site (IRES)-Cre fusion protein from the histidine decarboxylase (Hdc) promoter/enhancer elements. Hdc encodes the catalyzing enzyme involved in the formation of histamine which is released during allergic reactions and acts as a neurotransmitter in the brain. Histaminergic neurons regulate sleep-wake cycle. When Hdc is induced, cre recombinase is expressed in tuberomammillary nucleus (TMN) of the hypothalamus as well as other areas of the brain, mast cells, gastric mucosa, and fetal liver. When these mice are bred with mice containing loxP-flanked sequence, Cre-mediated recombination will result in deletion of the floxed sequences in the Hdc-expressing cells of the offspring. The donating investigator has not attempted to generate homozygous mice to date (March 2013).

    For example when bred to STOCK Gabrg2tm1Wul/J mice (Stock No. 021197), GABAA receptors in these GABAA receptor γ2 subunit deficient histaminergic neurons exhibit increased excitability with no effect on the sleep-wake cycle."

  • Hrh3−/− ( Mus musculus )

    "A neomycin selection cassette replaced a 0.7 kb fragment containing exon 1 and part of exon 2."

  • KA1R-Cre Tg ( Mus musculus )

    "Mice hemizygous for this "G32-4" transgene are viable, fertile, and do not display any gross physical or behavioral abnormalities. Transgene expression (cre activity) is detectable at 14 days old in area CA3 of the hippocampus, and at 8 weeks of age, recombination is observed in nearly 100% of pyramidal cells in area CA3. Recombination is also observed in other brain areas, but at distinctly lower frequencies. If bred with mice containing a loxP-flanked sequence of interest, tissue-specific deletion of that sequence results in the offspring. Specifically, when these mice were bred with a conditional CaMKII allele (see Stock No. 006575), the resulting offspring exhibited altered neurotransmitter release. The donating investigator reports that G32-4 females may confer global Cre-mediated deletion of loxP-flanked sequences in some offspring, and recommend using male G32-4 mice exclusively for such trials. These Cre-expressing mice may be useful in neurological studies involving hippocampus learning and memory, area CA3, and pyramidal cells."

    "The transgenic constuct consisted of the Grik4 promoter and exons 1 and 2 adjoined to the cre recombinase coding region. The Grik4 promoter was shown to drive expression in the CA3 pyramidal cell layer by staining of a mouse also harboring a lacZ reporter transgene that is expressed upon cre mediated recombination."

  • Nestin-Cre Tg ( Mus musculus )

    "These transgenic mice express Cre recombinase under the control of the rat nestin promoter and enhancer. Mice that are hemizygous for the transgenic insert are viable and fertile. Initial studies utilizing a reporter strain carrying a beta galactosidase transgene whose expression is dependent on Cre-mediated recombination indicate that cre is primarily expressed in the central and peripheral nervous system with a few isolated kidney and heart cells also expressing activity. The donating investigator indicates that Cre recombinase activity is present in nervous tissue by embryonic day 11. The transgene insertion location is on Chromosome 12, as determined by FISH analysis, view pdf."

    PDF is available from link below.

  • Nr3c1 loxP/loxP ( Mus musculus )

    "These mice possess loxP sites on either side of exon 2 of the targeted gene. Mice that are homozygous for this allele are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have exon 2 deleted in the cre-expressing tissue(s).

    When bred to a strain with Cre recombinase expression in the T cell lineage (see Stock No. 003802 for example), this mutant mouse strain may be useful in studies of hypothalamic-pituitary-adrenal axis homeostasis."

  • POMC-Cre Tg ( Mus musculus )

    "In this strain, the mouse pro-opiomelanocortin-alpha (Pomc) promoter drives expression of cre in the central nervous system, primarily the hippocampus. Cre expression is strongest and most restricted to the granule cells of the dentate gyrus subregion. Weaker, scattered expression can also be detected in other regions including the arcuate nucleus of the hypothalamus and the habenular nucleus. When crossed with a strain carrying a loxP-flanked genomic segment of interest, tissue-specific excision of that segment may be achieved.

    In POMC-cre and loxP-flanked (floxed) strain intercrosses, using male cre animals is recommended. According to the donating investigator, passing the cre through the female germline can result in a high percentage of offspring that carry the recombined allele in all of their tissues. Such "leaky" Cre expression is reported in male POMC-cre mice also, but at a lower frequency (< 5%). The frequency of "leaky" expression in male POMC-cre mice, however, increases with the animal's age."

  • Rosa26-floxed stop tdTomato ( Mus musculus )

    "Ai9 mice hemizygous for this Rosa-CAG-LSL-tdTomato-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream red fluorescent protein variant (tdTomato). When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissue(s); resulting in expression of tdTomato. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, tdTomato expression is determined by which tissue(s) express Cre recombinase. The donating investigator reports that Ai9 mice do not express tdTomato prior to introduction of Cre recombinase and tdTomato expression following exposure to cre can be detected by fluorescence and mRNA (in situ hybridization). Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired. These Ai9 mice are useful as a Cre reporter strain; expressing the red fluorescent protein variant, tdTomato, following Cre-mediated recombination.

    The Allen Institute for Brain Science (AIBS) website has specific characterization information for several Cre Driver and Cre Reporter lines. Please see their website for images of AIBS experiments performed with all lines.

    Of note, Ai9 mice may also be available on a C57BL/6J congenic background (see Stock No. 007909)."

  • Rosa26-floxed stop zsGreen ( Mus musculus )

    "Ai6 mice hemizygous for this Rosa-CAG-LSL-ZsGreen1-WPRE conditional allele are viable and fertile. A loxP-flanked STOP cassette prevents transcription of the downstream enhanced green fluorescent protein (ZsGreen1). When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissue(s); resulting in expression of ZsGreen1. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, ZsGreen1 expression is determined by which tissue(s) express Cre recombinase. The donating investigator reports that Ai6 mice do not express ZsGreen1 prior to introduction of Cre recombinase and ZsGreen1 expression following exposure to cre can be detected by fluorescence and mRNA (in situ hybridization). Bright fluorescence is observed mainly in cell bodies. Of note, the FRT sites flanking the mutation allow for additional targeted replacement of the reporter sequences through Flp-mediated recombination if so desired. Similarly, the attB/attP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase if so desired. These Ai6 mice are useful as a Cre reporter strain; expressing the enhanced green fluorescent protein, ZsGreen1, following Cre-mediated recombination.

    For characterization information, see images at the Allen Institute for Brain Science website (Ai6 images)."

  • tetO-tdTomato/SypGFP Tg ( Mus musculus )

    "Hemizygous TRE-Bi-SG-T line 1.1 transgenic mice are viable and fertile, with no reported phenotypic abnormalities. The TRE-Bi-SG-T transgene has Myc-tagged tdTomato and full-length mouse synaptophysin/mut4EGFP fusion protein (Syp-GFP) expression under the control of the bi-directional tet-responsive promoter (tetO or TRE). When bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), tdTomato and Syp-GFP fusion protein expression can be regulated with the tetracycline analog doxycycline (dox) in the resulting double mutant offspring. In tTA(-dox) or rtTA(+dox)-expressing cells, tdTomato expression is directed to the entire cell, while GFP expression is directed to the synapse/synaptic vesicle. The donating investigator reports that direct GFP fluorescence and direct tdTomato fluorescence may be visualized in these mice when tTA is present/dox is absent. In addition, the three Myc epitopes at the C-terminus of tdTomato allow fluorescent signal enhancement via immunofluorescence if needed. These TRE-Bi-SG-T transgenic mice are useful to generate mutant mice for Tet-Off/Tet-On and/or fluorescent protein applications.

    These TRE-Bi-SG-T transgenic mice may be used to design a tripartite system to express fluorescently tagged synaptic proteins with both spatial and temporal control. When bred with floxed-STOP-tTA mice (ROSA26-ZtTA; Stock No. 012266), the resulting double mutant mice allow tdTomato and Syp-GFP expression to be determined by the tissue-specific Cre recombinase expression of a third strain of the researchers choosing. For example, when TRE-Bi-SG-T transgenic mice are bred with floxed-STOP-tTA mice (ROSA26-ZtTA; Stock No. 012266) and a neural-specific Cre recombinase strain (for example: Foxg1-Cre; Stock Nos. 004337 or 006084), the resulting triple mutant mice should exhibit labeling of whole neurons in red and presynaptic terminals in green in the absence of dox."

  • Th loxP/loxP ( Mus musculus )

    "A loxP site was inserted 600 bp 5' of the transcription start site and a loxP site and FRT flanked neo cassette were inserted in intron 1 via homologous recombination. Flp mediated recombination removed the neo cassette."


Web Links:

Last updated: 2014-01-14T18:34:18.370-05:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016