C57BL/6-Tmem119^{em1(cre/ERT2)Gfng}/J

Stock number: 031820
Product name: Tmem119-2A-CreERT2
Research areas: Cancer Research, Endocrine Deficiency Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

Tmem119-2A-CreERT2 knock-in mice have the endogenous transmembrane protein 119 (Tmem119) promoter/enhancer sequences directing expression of tamoxifen-inducible Cre recombinase primarily in microglia, and are also designed to retain endogenous Tmem119 expression. These mice allow specific and inducible genetic manipulations in vivo for studying the function of resident microglia in the central nervous system during neuroinflammatory and neurological disease/injury, and may also be useful for investigating bone development, osteoblast differentiation and osteosarcoma.
Of note, the donating investigator has made the following TMEM119 alleles available from The Jackson Laboratory: Tmem119-2A-CreERT2 (Stock No. 031820) and Tmem119-2A-EGFP (Stock No. 031823).

Detailed description

Microglia are resident myeloid cells of the central nervous system (CNS), and become activated during neurological disease. Transmembrane protein 119 (TMEM119) is a highly-expressed microglia marker that can be useful for discriminating resident microglia from any infiltrating cell types (e.g., blood-derived macrophages) present in CNS injury/disease states. TMEM119 may also contribute to testis development, bone development and osteoblast differentiation, as well as function as an oncogene in osteosarcoma.

The Tmem119-2A-CreERT2 knock-in allele has a tamoxifen-inducible Cre recombinase fusion gene (CreER^T2) inserted into the stop codon of the transmembrane protein 119 locus (Tmem119). This is designed to have the endogenous Tmem119 promoter/enhancer driving CreERT2 expression primarily in microglia (see below), as well as to retain endogenous Tmem119 expression. Specific details are below.

Prior to tamoxifen exposure, mice heterozygous for this Tmem119-2A-CreERT2 knock-in allele are viable and fertile with normal breeding, and no reported gross phenotypic or behavioral abnormalities. Homozygous mice are viable and fertile. [February 2020]
Tmem119-2A-CreERT2 mice are expected to exhibit normal TMEM119 expression, although this has not yet been characterized. Furthermore, it has not yet been tested if any Cre recombinase activity occurs before tamoxifen. [August 2018]

When Tmem119-2A-CreERT2 mice are bred with mice containing lox-flanked (floxed) sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in Tmem119-expressing cells of the offspring. Specifically, the donating investigator reports tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Tmem119 expression pattern: CreERT2 expression is primarily in microglia, with some expression in non-microglial cell types (e.g., Iba1-negative leptomeningeal cells [fibroblast or fibroblast-like cells] residing in the pia and around some major blood vessels) - which may be due to some low level of Tmem119 expression in those cells.

Of note, the donating investigator has made the following TMEM119 alleles available from The Jackson Laboratory: Tmem119-2A-CreERT2 (Stock No. 031820) and Tmem119-2A-EGFP (Stock No. 031823).

The CreERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor that 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, CreERT2 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 co-administered.

Development

The Tmem119-2A-CreERT2 knock-in allele was created in the laboratory of Dr. Guoping Feng (Massachusetts Institute of Technology) using CRISPR/cas9 endonuclease-mediated genome editing.

First, a ribonucleoprotein complex (cas9-RNP) was created consisting of the S. pyogenes CRISPR/cas9 nuclease fused to a guide RNA sequence designed to target the stop codon of the transmembrane protein 119 locus (Tmem119) on chromosome 5. The cas9-RNP complex and a 2A-CreERT2 dsDNA (encoding a viral 2A oligopeptide that mediates ribosomal skipping and the CreER^T2 fusion gene [Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain]) with ~1.5 kbp homology arms were injected into the pronucleus of C57BL/6NTac embryos. Founder mice with the Tmem119-2A-CreERT2 allele were identified for correct 5' and 3' insertion by genotyping and fully sequenced. Founders were bred to C57BL/6J mice for germline transmission. The Tmem119-2A-CreERT2 colony was thereafter maintained by breeding together for several generations by the donating lab (see SNP note below). In 2018, Tmem119-2A-CreERT2 males with black coat color were sent to The Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

While the cas9-RNP approach is designed for efficient genome editing and significantly reduced off-target effects [Jacobi et al. 2017 Methods 121-122:16-28], the donating investigator reports the mice have not been specifically screened for the absence/presence of off-target mutations to date (August 2018).

In 2019 a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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. While the 43 markers throughout the genome suggested a C57BL/6 genetic background, three of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Tmem119^{em1(cre/ERT2)Gfng}
Name: endonuclease-mediated mutation 1, Guoping Feng
MGI accession ID: MGI:6195002
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible
Synonyms: Tmem119-CreERT2
Marker symbol: Tmem119
Marker name: transmembrane protein 119
Marker synonyms: OBIF; RGD1307573
Chromosome: 5
Strain of origin: C57BL/6NTac
Site of expression: The endogenous Tmem119 promoter/enhancer drives tamoxifen-inducible Cre recombinase (CreERT2) expression primarily in microglia. Specifically, the donating investigator reports tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Tmem119 expression pattern: CreERT2 expression is primarily in microglia, with some expression in non-microglial cell types (e.g., Iba1-negative leptomeningeal cells [fibroblast or fibroblast-like cells] residing in the pia and around some major blood vessels) - which may be due to some low level of Tmem119 expression in those cells.
Molecular note: The allele was created using CRISPR/cas9 endonuclease-mediated genome editing to target the stop codon of the transmembrane protein 119 locus (Tmem119) on chromosome 5. The cas9-RNP complex and a 2A-CreERT2 dsDNA (encoding a viral 2A oligopeptide that mediates ribosomal skipping and the CreER fusion gene with ~1.5 kbp homology arms were injected into the pronucleus of C57BL/6NTac mouse embryos. Founders were bred to C57BL/6J mice for germline transmission.