C57BL/6J-Cst7^{em3(folA/icre)Aduci}/J

Stock number: 040493
Product name: Cst7^DD-Cre
Strain synonyms: Cst7-DD-Cre
Research areas: Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

Cst7^DD-Cre knock-in mice are designed to have trimethoprim (TMP)-inducible (DD)-iCre-P2A sequence inserted immediately downstream of the translation initiation codon in exon 1 of the Cst7 gene. The endogenous Cst7 promoter directs TMP-inducible expression of DD-iCre recombinase to myeloid cells in the brain. These mice are useful for studying distinct signaling pathways and functions of disease associated microglia (DAMs) based on their location within the brain and their association with disease/injury by combining with appropriate floxed alleles.

Detailed description

The cystatin F (leukocystatin) (Cst7) gene encodes a glycosylated cysteine protease inhibitor that plays a role in immune regulation, inflammation, and cell death. Increased expression of this inflammatory gene is seen in microglia during their activation in response to development of Alzheimer's Disease (AD)-related pathology in mouse models of the disease, as well as in models of demyelination, as part of a disease associated microglial (DAM) response. DAMs are a subset of plaque associated microglia (PAMs) found in the brains of Alzheimer's disease (AD) patients where they have been characterized by their unique localization near Aβ plaques and their phagocytic and lipid-metabolizing features. Changes in gene expression and physical properties of microglia in the AD brain can occur in response to Aβ plaques, tau-laden neurons, or cell death and results in dramatic changes in microglial phenotypes.

Cst7^DD-Cre knock-in mice are designed to have a trimethoprim (TMP)-inducible icre recombinase gene followed by a 2A oligopeptide (P2A) self-cleaving peptide sequence inserted immediately downstream of the translation initiation codon in exon 1 of the Cst7 gene. The presence of DD-Cre-P2A cassette allows the expression of Cst7 and DD-Cre. DD-Cre is a TMP-inducible cre recombinase normally degraded through the proteasomal pathway. However, in the presence of TMP, the DD-Cre fusion protein is stabilized and does not degrade, enabling long-term and temporal recombination at loxP sites. TMP is safer for mice compared to Tamoxifen and can be added to drinking water allowing for long-term treatment, unlike Tamoxifen which can only safely be administered short-term. Homozygous mice are viable and fertile. When these mice are bred with mice containing loxP-flanked sequence, iCre-mediated recombination will result in deletion of the loxP-flanked sequences in myeloid cells in the brain.

When crossed to 5xFAD mice and Ai14^tdTomato mice (Stock No. 007914), 2.5 month old and 9.5-month-old 5xFAD hemizygous, double heterozygous Cst7^DD-Cre Ai14^tdTomato mice exhibit tdTomato expression in DAMs surrounding plaques, while none was found in any other microglial populations (including homoeostatic), nor in any other cell types in the brain, after mice were treated with trimethoprim for 1.5 months.

Of note, Cst7^Cre mice are a similar CRISPR-generated knock-in of a P2A-iCre immediately downstream of the translation initiation codon in exon 1 of the Cst7 gene and is available as Stock No. 040492. Cst7^Cre mice are suitable for robust transient myeloid cell manipulation making them unsuitable for DAM targeting, but useful for targeting the majority of brain macrophage populations.

Development

sgRNAs (TMF1131-CCTGCATTTCCCCAGCCATG and TMF-1132-AAGCAGAATGGCCAGCCACA) were used to target sequences immediately downstream of the translation initiation codon in exon 1 of the cystatin F (leukocystatin) (Cst7) gene for insertion of a 1596 bp DNA sequence encoding a mutant destabilized domain from bacterial dihydrofolate reductase (ecDHFR) fused to an icre recombinase gene followed by a 2A oligopeptide (P2A) self-cleaving peptide, to mediate ribosomal skipping. sgRNA/tracrRNA ribonucleoprotein complex (RNP), CAS9 protein, and the 1596 base ssODN sequence was introduced into the cytoplasm of fertilized eggs with well recognized pronuclei from C57BL/6J mice. Embryos were transferred to pseudopregnant CD1 females. Correctly targeted Cst7^DD-Cre pups were identified by PCR and sequencing were further bred to C57BL/6J mice (Stock No. 000664) for at least 2 generations to remove off-target sites. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Cst7^{em3(folA/icre)Aduci}
Name: endonuclease-mediated mutation 3, Frank LaFerla
MGI accession ID: MGI:8163419
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Synonyms: Cst7^DD-Cre; Cst7^{em3(folA/cre)Aduci}
Marker symbol: Cst7
Marker name: cystatin F (leukocystatin)
Marker synonyms: CMAP; cystatin-like metastasis-associated protein; Leukocystatin
Chromosome: 2
Strain of origin: C57BL/6J
Molecular note: sgRNAs (TMF1131-CCTGCATTTCCCCAGCCATG and TMF-1132-AAGCAGAATGGCCAGCCACA) were used to target sequences immediately downstream of the translation initiation codon in exon 1 of the cystatin F (leukocystatin) (Cst7) gene for insertion of a 1596 bp DNA sequence encoding a mutant destabilized domain from bacterial dihydrofolate reductase (ecDHFR) fused to an icre (codon optimized cre) recombinase gene followed by a 2A oligopeptide (P2A) self-cleaving peptide, to mediate ribosomal skipping.