B6;FVB-Tg(Aldh1l1-cre/ERT2)1Khakh/J

Stock number: 029655
Product name: Aldh1l1-Cre/ERT2 BAC transgenic
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Aldh1l1-Cre/ERT2 BAC transgenic mice have tamoxifen-inducible Cre recombinase expression directed at high levels to the vast majority of astrocytes, with no detectable expression in neurons. These mice allow pan-astrocytic, specific and inducible genetic manipulations in vivo for studying astrocyte biology at different developmental stages in neural circuitry/synapses, behavior, disease and injury/trauma.

Aldh1l1-Cre/ERT2 BAC transgenic mice are also available on a C57BL/6N-congenic background (Stock No. 031008).

Detailed description

Astrocytes exist throughout the nervous system and are proposed to function in behavior, neural circuitry, synapse formation/regulation, calcium signaling in locomotion/startle responses and disease.

The ~230.5 kbp Aldh1l1-Cre/ERT2 BAC transgene has tamoxifen-inducible Cre recombinase expression directed to astrocytes (but not neurons) by the aldehyde dehydrogenase 1 family member L1 promoter/enhancer regions. When Aldh1l1-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 Aldh1l1-expressing cells of the offspring.
Specifically, tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Aldh1l1 expression pattern. The donating investigator summarizes that Aldh1l1-Cre/ERT2 mice have very high CreER^T2 expression in the vast majority of astrocytes, with no detectable expression in neurons. Cre recombinase expression in other tissues has not been investigated to date (October 2016). These mice allow temporal control of pan-astrocytic, Cre-lox genetic manipulations in vivo, including adult mice.
For example, when Aldh1l1-Cre/ERT2 BAC transgenic mice are bred to have the Cre-inducible membrane-tethered GCaMP6f allele (Lck-GCaMP6f^flox ; Stock No. 029626) or the Cre-inducible cytosolic GCaMP6f allele (Ai95 ; Stock Nos. 024105/028865), the resulting double mutant mice show ubiquitous and high GCaMP6f expression (green fluorescence) in a vast majority (>95%) of astrocytes in the hippocampal CA1, CA3 and DG regions and in V1 of the visual cortex - all absent of GCaMP6f expression in neurons. Both double mutant lines allow measurement of astrocyte calcium signaling (evoked and spontaneous) using brain slice imaging or live behaving mice, and highlight features of Lck-GCaMP6f that are separable/distinct from those reported for Ai95 (e.g., membrane-tethered Lck-GCaMP6f labels the entire surface area of astrocytes, whereas cyto-GCaMP6f most clearly revealed the internal volumes).
In addition, Aldh1l1-Cre/ERT2 BAC transgene expression has no reported alterations on astrocyte morphology. Following injection of Cre-dependent GFP adenovirus and subsequent tamoxifen treatment, Aldh1l1-Cre/ERT2 BAC transgenic mice exhibit GFP-labeled astrocytes with typical, unambiguous, complex bushy astrocyte morphologies.
To determine the cortical astrocyte transcriptome in fully developed adult mice, Aldh1l1-Cre/ERT2 BAC transgenic mice may be bred to have Cre-dependent expression of hemagglutinin epitope-tagged ribosomes (RiboTag ; see Stock No. 011029). The resulting Aldh1l1-Cre/ERT2;RiboTag mice allow the exploration of astrocyte diversity between specific adult neural circuits and brain areas.

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 ALDH1L1 from the BAC Aldh1l1 locus, the ALDH1L1 RNA/protein levels between Aldh1l1-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

Aldh1l1-Cre/ERT2 BAC transgenic mice were designed in the laboratory of Dr. Baljit S. Khakh (University of California Los Angeles).
The ~228 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-7M9 was obtained; containing the entire aldehyde dehydrogenase 1 family member L1 locus (Aldh1l1), as well as ~49 kbp 5' (centromeric) flanking region and ~130 kbp 3' (telomeric) flanking region. To date (October 2016), no other genes are known to exist in the centromeric sequences, and the telomeric region includes one complete locus (Slc41a3) and one incomplete locus (a ~70 kbp 3' portion of Iqsec1). 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 Aldh1l1 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 ~230.5 kbp Aldh1l1-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 two founder lines with germline transmission. Founder line #1 was established and characterized. The donating investigator reports that Aldh1l1-Cre/ERT2 BAC transgenic mice from this founder line were backcrossed several 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. The live colony was rederived using one round of breeding to C57BL/6J (Stock No. 000664). The first generation rederived mice were bred once to C57BL/6NJ (Stock No. 005304). The resulting colony was then maintained by breeding Aldh1l1-Cre/ERT2 BAC transgenic mice to wildtype (noncarrier) mice.
Of note, the transgene insertion site(s) and copy number are not known to date (October 2016).

In 2016, a SNP (single nucleotide polymorphism) panel analysis, with markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. This revealed ~25% of the markers were not fixed for C57BL/6 allele-type (i.e., still segregating for FVB/N allele-type markers [seven different chromosomes]). Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were incompletely backcrossed onto C57BL/6.

By August 2017, SNP analysis and breeding history suggest our live colony is ~88-94% C57BL/6 [C57BL/6N:C57BL/6J ratio 2:1] and ~6-12% FVB/N.

Allele

Symbol: Tg(Aldh1l1-cre/ERT2)1Khakh
Name: transgene insertion 1, Baljit Khakh
MGI accession ID: MGI:5806568
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Tg(Aldh1l1-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: Tamoxifen-inducible Cre recombinase activity in these mice recapitulates the endogenous Aldh1l1 expression pattern in the vast majority of astrocytes.
Molecular note: The transgene contains the entire containing the entire aldehyde dehydrogenase 1 family member L1 locus (Aldh1l1), as well as ~49 kbp 5' (centromeric) flanking region and ~130 kbp 3' (telomeric) flanking region from BAC RP23-7M9. Using homologous recombination/BAC recombineering, a CreER cDNA sequence followed by an SV40 polyadenylation signal was inserted into exon 1 of the BAC Aldh1l1 gene immediately preceding the translation initiation codon. No other loci on the BAC were altered. Two founder lines with germline transmission are generated. Founder line #1 was established and characterized.