FVB.Cg-Tg(tetO/Prnp-GLRA1*,-EYFP)9518Jjan/J

Stock number: 029299
Product name: TRE-GlyCl line 9518
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

TRE-GlyCl transgenic mice are a chemogenetic tool allowing tet-controllable, non-invasive neuronal silencing and in vivo cellular electrical activity inhibition. They have tet-conditional (inducible/reversible) expression of EYFP and GlyClα^F207A/A288G; a glycine-insensitive, mutant human glycine α1 receptor that induces a hyperpolarizing chloride current specifically following administration of ivermectin (IVM; a well-tolerated, inexpensive and widely-available anti-parasitic compound). Several founder lines are available; each with an unique expression pattern.

Detailed description

TRE-GlyCl transgenic mice are a chemogenetic tool allowing non-invasive neuronal silencing and in vivo cellular electrical activity inhibition. The TRE-GlyCl transgene has a third generation Tet response element promoter (P_TRE3G) and mouse prion protein promoter sequences directing tricistronic expression of individual GlyClα^F207A/A288G and EYFP proteins.

Glycine receptor chloride channels (GlyCl) are ligand-gated ion channels that, upon binding their native neurotransmitter glycine, produce a hyperpolarizing chloride current that curtails the initiation of action potentials in mature neurons (e.g., neuronal silencing). GlyCl functions efficiently as an α subunit homopentamer with large single channel conductance (90 pS). GlyClα^F207A/A288G is a mutant human glycine α1 receptor with two amino acid substitutions (F207A and A288G) that diminish glycine sensitivity while elevating ivermectin (IVM) sensitivity. IVM is a well-tolerated, inexpensive and widely-available anti-parasitic compound that reaches the brain following systemic administration.

When TRE-GlyCl transgenic mice are bred with another mouse expressing tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), expression of GlyClα^F207A/A288G and EYFP can be regulated with tetracycline or its analog doxycycline (dox). Cells expressing both TRE-GlyCl and tTA(-dox) [or rtTA(+dox)] can be identified by EFYP labeling and can be hyperpolarized following IVM administration. EYFP is observed by direct fluorescence, and antibody immunostaining may be needed to enhance signal for some applications. For example, when bred to CaMKIIα-tTA transgenic mice (Stock No. 003010), the resulting bigenic CaMKII-GlyCl mice allow forebrain neuron silencing that can be regulated by dox and IVM.


Although TRE-GlyCl transgenic mice should not express GlyCl or EYFP independent of tTA/rtTA, cerebellar EYFP expression is observed without the transactivator in several TRE-GlyCl lines. As such, it is recommended that TRE-GlyCl single transgenic animals (with no tTA/rtTA) be included as controls in all experiments.

The donating investigator reports that mice hemizygous for the TRE-GlyCl transgene are viable and fertile with no observed behavioral abnormalities. To date (June 2016), it has not been attempted to make this strain homozygous.

Several TRE-GlyCl transgenic founder lines are available, each with an unique expression pattern. The pattern of EYFP expression for each line is summarized below, both for single TRE-GlyCl transgenic mice (tTA-independent; using antibody immunostaining) and for bigenic CaMKII-GlyCl mice (tTA-driven; via direct fluorescence). Overall, lines 9531 and 9542 displayed the strongest signal. Line 9531 is recommended by the donating investigator for most experimental purposes as it displayed less tTA-independent expression in the cerebellum than line 9542. Lines 9487 and 9511 displayed the weakest overall signal.

TRE-GlyCl line 9487 (Stock No. 029296):
  tTA-independent: sparse labeling in cerebellar granule cell layer of vermis lobules 1-3, as well as hemisphere lobules, copula pyramidus and paramedian.
  CaMKII-GlyCl(9487) bigenic: sparse scattered labeling throughout neocortex, limited striatal expression; cortical labeling was stronger in superficial layers. Lines 9487 and 9511 displayed the weakest overall signal.

TRE-GlyCl line 9492 (Stock No. 029297):
  tTA-independent: sparse cell labeling of molecular layer throughout cerebellar cortex, possibly basket cells.
  CaMKII-GlyCl(9492) bigenic: dense neocortical and hippocampal labeling with scattered striatal expression of consistent moderate signal intensity; cortical expression spanned all layers; hippocampal labeling was dense in CA1, mosaic in the dentate and absent from CA3.

TRE-GlyCl line 9511 (Stock No. 029298):
  tTA-independent: strong dense labeling throughout all lobules of the cerebellar cortex, primarily localized to granule cell bodies and their axonal projections within the molecular layer, consistent anterior-posterior labeling throughout the cerebellum; absent from Purkinje neurons.
  CaMKII-GlyCl(9511) bigenic: sparse labeling of varying intensity throughout neocortex, hippocampus and striatum. Lines 9487 and 9511 displayed the weakest overall signal.

TRE-GlyCl line 9518 (Stock No. 029299):
  tTA-independent: faint but dense labeling in cerebellar granule cell layer of central vermis and associated simplex; strong dense labeling of granule cell bodies and their axonal projections in cerebellar hemispheres; absent from Purkinje neurons.
  CaMKII-GlyCl(9518) bigenic: scattered neocortical and striatal labeling with limited hippocampal expression of low-moderate signal intensity.

TRE-GlyCl line 9522 (Stock No. 029300):
  tTA-independent: faint labeling of pyramidal neurons throughout neocortex, somewhat stronger in layers 2/3 than 5; similar faint labeling of neuronal cell body layers in all hippocampal subfields.
  CaMKII-GlyCl(9522) bigenic: dense neocortical, hippocampal and striatal labeling, consistent low-moderate signal intensity; cortical expression spanned all layers; hippocampal labeling was dense in CA1, mosaic in the dentate and absent from CA3.

TRE-GlyCl line 9531 (Stock No. 029301):
  tTA-independent: some animals display sparse but intense labeling within the granule cell layer proximate to the Purkinje cell layer, occasional cells additionally located in molecular layer, labeling distributed across all cerebellar lobules.
  CaMKII-GlyCl(9531) bigenic: dense neocortical, hippocampal and striatal labeling of varying intensity; cortical expression spanned all layers, somewhat higher intensity rostral than caudal; hippocampal labeling was mosaic in CA1 and dentate, absent from CA3. Lines 9531 and 9542 displayed the strongest overall signal. Line 9531 is recommended by the donating investigator for most experimental purposes as it displayed less tTA-independent expression in the cerebellum than line 9542.
  Additionally, breeding TRE-GlyCl line 9531 to harbor the Nop/Klk8-tTA transgene generates Nop/Klk8-GlyCl(9531) bigenic mice with EYFP expression in the entorhinal cortex. Following IVM treatment, those mice exhibit impaired associative memory formation in fear conditioning.

TRE-GlyCl line 9542 (Stock No. 029302):
  tTA-independent: faint sparse labeling in cerebellar granule cell layer of central vermis and associated simplex becoming more dense anteriorly; strong dense labeling of granule neurons in both cerebellar hemispheres.
  CaMKII-GlyCl(9542) bigenic: dense neocortical and striatal labeling of some varying intensity; cortical expression spanned all layers, somewhat more dense caudal than rostral; hippocampal labeling was mosaic in CA1 and dentate, sparse in CA3. Lines 9531 and 9542 displayed the strongest overall signal. Line 9531 is recommended by the donating investigator for most experimental purposes as it displayed less tTA-independent expression in the cerebellum than line 9542.

The primary publication (Zhao et al. 2016 Cell Rep) protocol for IVM is summarized below:
For all in vivo experiments, Ivomec (1% ivermectin injectable solution, Merial U.S.) was diluted to 0.2% with vehicle (3:2 mix of propylene glycol and 0.9% saline). Stock dilutions were made fresh on the day of use and administered via intraperitoneal injection at 5 mg/kg. For in vitro electrophysiology studies, Ivermectin powder (Sigma I8898) was dissolved in DMSO at a stock concentration of 100 μM, stored at -20º C for up to 1 week, and diluted daily to 100 nM with ACSF for the final working solution.

Development

The TRE-GlyCl transgene (moPrp-TRE3G-GlyClα-2A-YFP-2A-GlyClα-WPRE) was designed by Dr. Joanna L. Jankowsky (Baylor College of Medicine). The transgene has a Tet-responsive element 3G promoter and the mouse prion protein non-coding exons 1-2 upstream of a GlyClα^F207A/A288G::2A::EYFP::2A::GlyClα^F207A/A288G coding cassette (described below), followed by the mouse prion protein non-coding exon 3 and the woodchuck hepatitis post-transcriptional regulatory element (WPRE).


The construct uses the third generation tetracycline response element promoter P_TRE3G (bp 7-382; containing seven copies of the 19-bp tet operator sequence [tetO] followed by the minimal human cytomegalovirus promoter). The P_TRE3G promoter in this construct replaces the tetO promoter in the original moPrP.XhoI vector).


The GlyClα^F207A/A288G::2A::EYFP::2A::GlyClα^F207A/A288G cassette has an enhanced yellow fluorescent reporter (EYFP) flanked on either side by a human glycine receptor α1 (GLRA1) cDNA sequence harboring the point mutations F207A (to eliminate endogenous ligand [glycine] sensitivity) and A288G (to increase ivermectin sensitivity almost 100-fold). To achieve tricistronic expression of the individual proteins, they are separated by 2A peptide sequences (from Thosea asigna virus).


The final 10.9 kbp transgene (of which the TRE-GlyCl sequences are 9.3 kbp) was injected into the pronucleus of B6C3F2 embryos. Founder mice were test-mated to C57BL/6J-congenic CaMKIIα-tTA transgenic mice (derived from Stock No. 003010) to assess the intensity/stability of EYFP expression across the forebrain as well as the responsiveness/sensitivity to ivermectin. Once identified to have desired expression and germline capability, the founder male #9518 was bred to FVB/NJ females to establish founder line 9518. The TRE-GlyCl line 9518 colony was backcrossed with FVB/NJ mice for a total of six generations prior to sending hemizygous males with albino coat color to The Jackson Laboratory Repository in 2016.

Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize oocytes from FVB/NJ female mice (Stock No. 001800). The TRE-GlyCl line 9518 colony was then maintained by breeding transgenic mice together, to wildtype (noncarrier) mice from the colony or to FVB/NJ inbred mice.

Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a FVB/NJ inbred male (thus the Y chromosome of the congenic strain is of FVB/NJ origin). The chromosomal insertion site(s) and transgene copy number has not been identified to date (June 2016).

Allele

Symbol: Tg(tetO/Prnp-GLRA1*,-EYFP)9518Jjan
Name: transgene insertion 9518, Joanna L Jankowsky
MGI accession ID: MGI:5779811
Generation method: Transgenic
Attributes: Reporter; Inducible; Humanized sequence
Marker symbol: Tg(tetO/Prnp-GLRA1*,-EYFP)9518Jjan
Marker name: transgene insertion 9518, Joanna L Jankowsky
Chromosome: UN
Strain of origin: (C57BL/6J x C3H/HeJ)F2
Expressed genes: GLRA1,glycine receptor alpha 1,human; YFP,Yellow Fluorescent Protein,jellyfish
Site of expression: Brain.
Molecular note: The transgene has a Tet-responsive element 3G promoter and the mouse prion protein non-coding exons 1-2 upstream of a cassette containing an enhanced yellow fluorescent reporter (EYFP) flanked on either side by a human glycine receptor alpha1 (GLRA1) cDNA sequence harboring the point mutations F207A (to eliminate endogenous ligand [glycine] sensitivity) and A288G (to increase ivermectin sensitivity almost 100-fold), followed by the mouse prion protein non-coding exon 3 and the woodchuck hepatitis post-transcriptional regulatory element (WPRE). To achieve tricistronic expression of the point mutations and EYFP, they are separated by T2A peptide sequences (from Thosea asigna virus).