B6.129S4(Cg)-Kcnn2^tm2Jpad/J

Stock number: 009602
Product name: SK2T
Research areas: Neurobiology Research, Research Tools

Description

The SK2^T mutant allele results in approximately ten-fold overexpression of the Kcnn2 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2; also called SK2) gene product prior to administration of tetracycline (or its analog doxycycline (dox)). These SK2-tTA mice may be useful in studying the role of small-conductance calcium-activated potassium (SK) channels in after-hyperpolarization and action potentials in neurobiology, hippocampal synaptic plasticity, learning and memory, and fear conditioning.

Detailed description

The SK2^T mutant allele has a tetracycline-based genetic switch inserted into the 5' UTR of the targeted gene, just upstream of the translation initiation site. This genetic switch harbors both the tetracycline-controlled transactivator protein (tTA) as well as the tetracycline operator (tetO; also called tetracycline-responsive element (TRE) or tet-operator). The donating investigator reports that long term administration of tetracycline (or its analog doxycycline (dox)) does not block transcription of the downstream Kcnn2 locus. The donating investigator also reports that homozygous mice are viable but do not thrive without dox treatment. SK2-tTA heterozygotes (SK2^T) exhibit approximately ten-fold overexpression of SK2 protein before dox treatment. In the absence of dox, homozygous mice exhibit enhanced SK channel-mediated restriction of glutamatergic activity in CA1 neurons, attenuated hippocampal synaptic plasticity, impaired spatial learning and memory in the Morris water maze, and impaired contextual and cued fear conditioning.

Development

A targeting construct was designed with (from 5' to 3') an adenovirus tripartite leader sequence, a binary tetracycline controlled transactivator (tTA) protein, an SV40 polyA signal, a loxP-flanked selection cassette (neomycin resistance and yeast URA3 gene), an hGH polyA signal, and 5 copies of the tetracycline operator (tetO; also called tetracycline-responsive element (TRE) or tet-operator) sequence fused to the cytomegalovirus minimal promoter. This construct was designed to insert 40 bp upstream of the most 5' methionine codon of the targeted gene. The construct was electroporated into 129/SvJae-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. To remove the floxed selection cassette, chimeric mice were bred to CMV-Cre mice (reported to be C57BL/6J genetic background; see Stock No. 006054). The donating investigator reports that heterozygous (SK2-tTA or SK2^T) mice were subsequently backcrossed for approximately 20 generations to C57BL/6J mice prior to arrival at The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Kcnn2^tm2Jpad
Name: targeted mutation 2, John P Adelman
MGI accession ID: MGI:3522587
Generation method: Targeted
Attributes: Inserted expressed sequence
Marker symbol: Kcnn2
Marker name: potassium intermediate/small conductance calcium-activated channel, subfamily N, member 2
Chromosome: 18
Strain of origin: 129S4/SvJae
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli
Site of expression: In the absence of dox, homozygous mice exhibit enhanced SK channel-mediated restriction of glutamatergic activity in CA1 neurons, attenuated hippocampal synaptic plasticity, impaired spatial learning and memory in the Morris water maze, and impaired contextual and cued fear conditioning.
Molecular note: A genetic switch based on the tetracycline transactivator/tetracycline operator (tTA/tetO) system was inserted into the 5' untranslated region (5' UTR) of the targeted gene 40 nucleotides upstream of the initiator ATG. The switch, after in vivo excision of a selection cassette, comprises a tripartite adenovirus leader sequence (to enhance translation initiation efficiency) followed by the coding sequence for the tTA protein with SV40 and human growth hormone polyadenylation signals separated by a single loxP site; and five copies of the tetracycline operator (tetO), also called the tetracycline responsive element (TRE), joined to the cytomegalovirus (CMV) minimal promoter. In the absence of doxycycline (Dox), heterozygous mutant mice express 4.5-fold the wild-type level of Kcnn2 mRNA and 10-fold the wild-type level of Kcnn2 subunit protein, respectively assessed by quantitative and real-time PCR of whole-brain RNA and by immunoblot analysis of membrane proteins from whole brains. Administration of Dox reduces, but does not eliminate, expression from the mutant allele.