B6N.FVB-Tg(Slc6a11-cre/ERT2)1Khakh/J

Stock number: 029656
Product name: Slc6a11-Cre/ERT2 BAC transgenic (GAT3-Cre/ERT2)
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Slc6a11-Cre/ERT2 BAC transgenic mice have tamoxifen-inducible Cre recombinase expression directed at moderate levels to astrocytes and neurons. These mice allow inducible genetic manipulations in vivo for studying astrocyte and neuron biology of GAT3+ cells.

Detailed description

The solute carrier family 6 (neurotransmitter transporter GABA) member 11 gene (Slc6a11) encodes GAT3 (Gabt4), a sodium-dependent transporter that uptakes the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), which ends the GABA neurotransmission. Defects in Slc6a11 may result in epilepsy or behavioral and intellectual defects.

The ~205.5 kbp Slc6a11-Cre/ERT2 BAC transgene has tamoxifen-inducible Cre recombinase expression directed to astrocytes and neurons by the Slc6a11 promoter/enhancer regions. When Slc6a11-Cre/ERT2 transgenic mice are bred with mice containing lox-flanked (floxed) sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in Slc6a11-expressing cells of the offspring.
Specifically, tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Slc6a11 expression pattern. The donating investigator summarizes that Slc6a11-Cre/ERT2 mice have moderate levels of CreER^T2 expression in astrocytes and neurons. Cre recombinase expression in other tissues has not been investigated to date (October 2016). These mice allow temporal control of Cre-lox genetic manipulations in vivo, including adult mice.
Following injection of Cre-dependent GFP adenovirus and subsequent tamoxifen treatment, Slc6a11-Cre/ERT2 BAC transgenic mice exhibit GFP-labeled astrocytes and neurons with typical morphologies.

The donating investigator reports there is no Cre recombinase activity prior to tamoxifen exposure. While the insertion of Cre/ERT2 should disable transcription/translation of SLC6A11 from the BAC Slc6a11 locus, the SLC6A11 RNA/protein levels between Slc6a11-Cre/ERT2 BAC transgenic mice and wildtype (noncarrier) mice was not characterized. Hemizygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. To date (October 2016), it has not been attempted to make this strain homozygous.

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

Slc6a11-Cre/ERT2 BAC transgenic mice were designed in the laboratory of Dr. Baljit S. Khakh (University of California Los Angeles).
The ~203 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-470B21 was obtained; containing the entire solute carrier family 6 (neurotransmitter transporter GABA) member 11 locus (Slc6a11 [GAT3 or Gabt4]), as well as ~48 kbp 5' (centromeric) flanking region and ~36 kbp 3' (telomeric) flanking region. To date (October 2016), no other confirmed genes are known to exist in the flanking regions, and the telomeric region includes an incomplete locus (an ~3.4 kbp 5' portion of Slc6a1 [GAT1 or Gabt1]). Using homologous recombination/BAC recombineering, a CreER^T2 cDNA sequence (Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain) followed by an SV40 polyadenylation signal was inserted into exon 1 of the BAC Slc6a11 gene (immediately preceding the translation initiation codon). No other loci on the BAC were altered. The CreER^T2 sequence harbors a rare AsiS1 restriction site (cutting with AsiS1 linearizes the BAC transgene). The final ~205.5 kbp Slc6a11-Cre/ERT2 BAC transgene was purified and then used for pronuclear injection into FVB/N oocytes fertilized by FVB/N sperm. Founder animals were bred to C57BL/6NTac inbred mice, generating founder lines with germline transmission. A founder line (#1) was established and characterized. The donating investigator reports that Slc6a11-Cre/ERT2 BAC transgenic mice from this founder line were backcrossed at least eight generations to C57BL/6NTac inbred mice (see SNP results below), and then hemizygous males with black coat color were sent to The Jackson Laboratory Repository in 2016. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).
Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6N inbred male (thus the Y chromosome of the congenic strain is of C57BL/6N origin). In addition, the transgene insertion site(s) and copy number are not known to date (October 2016).

In 2016-2017, SNP (single nucleotide polymorphism) panel analyses, with markers covering all 19 chromosomes and the X chromosome, as well as markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository and our first generation rederived living colony. The markers show our live colony to be ~96% C57BL/6 allele-type, with all C57BL/6 substrain markers tested as C57BL/6N allele-type. Some transgenic mice had two markers segregating for C57BL/6 or FVB/N allele-type - one marker each on chromosomes 7 (~36 Mbp) and 9 (~44 Mbp).

Allele

Symbol: Tg(Slc6a11-cre/ERT2)1Khakh
Name: transgene insertion 1, Baljit Khakh
MGI accession ID: MGI:5806574
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Tg(Slc6a11-cre/ERT2)1Khakh
Marker name: transgene insertion 1, Baljit Khakh
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: These mice have moderate levels of cre/ERT2 expression in astrocytes and neurons.
Molecular note: The transgene contains the entire solute carrier family 6 (neurotransmitter transporter GABA) member 11 locus (Slc6a11 [GAT3 or Gabt4]), as well as ~48 kbp 5' (centromeric) flanking region and ~36 kbp 3' (telomeric) flanking region from BAC RP23-470B21. Using homologous recombination/BAC recombineering, a CreER cDNA sequence followed by an SV40 polyadenylation signal was inserted into exon 1 of the BAC Slc6a11 gene immediately preceding the translation initiation codon. No other loci on the BAC were altered. Unstated number of founder lines with germline transmission are generated. A founder line #1 was established and characterized.