C57BL/6J-Slc47a1^{em1(cre/ERT2)Jkpns}/J

Stock number: 039218
Product name: Slc47a1-creER^T2
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

Slc47a1-creER^T2 knock-in mice have cre-ERT2-P2A inserted between exon 16 and the 3' UTR of the Slc47a1 gene. They are useful for applications requiring tamoxifen-induced deletion of floxed sequences in dural border cells (DBCs) of the brain and when studying the blood-cerebrospinal fluid barrier.

Detailed description

CRISPR/Cas9 generated Slc47a1-creER^T2 knock-in mice have cre-ERT2-P2A inserted between exon 16 and the 3' UTR of the solute carrier family 47, member 1 (Slc47a1) gene. The insertion of cre-ERT2-P2A does not affect endogenous gene expression. These mice express cre-ERT2 in putative dural border cells (DBCs). The dural border cell layer is positioned immediately above the arachnoid barrier cell (ABC) layer and forms a B-CSFB between the dura mater and cerebrospinal fluid (CSF) in the subarachnoid space (SAS). Restricted to the cytoplasm, cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. Heterozygotes are viable and fertile with no overt phenotype. The donating investigator did not maintain a homozygous colony, it is unknown if homozygotes are viable and fertile.

When these mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the Cre-expressing cells of the offspring.
For example, when bred to Ai6(RCL-ZsGreen) mice (Stock No. 007906), recombination is evident in in the leptomeninges in irregularly shaped cells, which sit above the ABC layer in the expected location of DBCs, after tamoxifen induction. No cre activity is detected prior to tamoxifen administration in the brain/dura.

The donating investigator also made a similar mouse line, Dpp4-creERT2 mice (Stock No. 039217), which express cre/ERT2 in arachnoid barrier cells (ABCs) of the brain.

Development

sgRNAs (TATATCAGAATTGAATAATGNGG and ATATCAGAATTGAATAATGTNGG) were designed to insert a cre/ERT2 fusion gene (a Cre recombinase fused to a human estrogen receptor ligand binding domain) fused to a P2A self-cleaving peptide between exon 16 and the 3' UTR of the solute carrier family 47, member 1 (Slc47a1) gene. sgRNA, the cas9 protein, and a donor plasmid were introduced into the cytoplasm of C57BL/6J-derived fertilized eggs with well-recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Slc47a1-creER^T2 mice were identified by PCR and Southern blot, and random insertions were removed. Mice were further backcrossed to C57BL/6J mice (Stock No. 000664) for at least 4 generations to develop the colony. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Slc47a1^{em1(cre/ERT2)Jkpns}
Name: endonuclease-mediated mutation 1, Jonathan Kipnis
MGI accession ID: MGI:7595999
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Synonyms: Slc47a1-creER^T2
Marker symbol: Slc47a1
Marker name: solute carrier family 47, member 1
Marker synonyms: 1300013J15Rik; MATE1; MATE2; MATE2-B; MATE2K; MATE2-K; mMATE1; mMATE1b; RGD1311123
Chromosome: 11
Strain of origin: C57BL/6J
Molecular note: Using CRISPR/Cas9 technology, a cre/ERT2 fusion gene with a P2A self-cleaving peptide were inserted between the exon 16 and the 3' UTR of the Slc47a1 gene.