C57BL/6N-Il1r1^tm1Quan/J

Stock number: 024101
Product name: IL-1R1 restore knock-in (IL-1R1^r)
Research areas: Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

The interleukin 1 type I receptor restore allele (IL-1R1^r) is a functional null prior to Cre recombinase exposure. These mice allow Cre recombinase-inducible restoration of functional IL-1R1 expression, and visualization of IL-1R1-expressing cells with concordant labeling of IL-1R1 mRNA (by tdTomato fluorescence) and protein (by 3xHA epitope). IL-1R1^r mice may be useful for studying cell-type-specific functions of IL-1R1 in neuroimmunity and neuroinflammation.

Detailed description

The interleukin 1 type I receptor restore allele (IL-1R1^r) has a floxed interfering DNA region (comprised of three disrupting elements) preventing transcription/translation of IL-1R1 prior to Cre recombinase exposure, and has sequences inserted in place of the endogenous stop codon that allow tracking of Cre recombinase-restored expression of IL-1R1 mRNA (tdTomato fluorescence) and protein (3xHA epitope).

Homozygous (IL-1R1^r/r) mice have no endogenous IL-1R1 mRNA expression in any tissues, and exhibit the expected IL-1R1 knockout phenotype (no IL-1-induced CNS effects). Specifically, intracerebroventricular injection of IL-1β into IL-1R1^r/r mice did not produce brain endothelial COX-2 expression, CNS leukocyte infiltration or global microglia activation.

Prior to exposure to Cre recombinase, no tdTomato fluorescence or 3xHA expression is detected in brain tissue of IL-1R1^r/r mice.


Following Cre recombinase exposure, the resulting IL-1R1 Restored allele (IL-1R1^R) expresses functional IL-1R1, with robust tdTomato fluorescence in the cytoplasm of IL-1R1 mRNA-producing cells and 3xHA epitope-tagged IL-1R1 protein anchored to the cell membrane in the same cells. The restored 3xHA epitope-tagged IL-1R1 protein is readily detected by IHC in the normal brain.

The donating investigator reports that IL-1R1^r/r mice are viable and fertile with normal lifespan and no other gross abnormalities.

Development

The interleukin 1 type I receptor restore allele (IL-1R1^r) was created in the laboratory of Dr. Ning Quan (The Ohio State University) by inserting two knock-in target sequences into the interleukin 1 receptor, type I gene (Il1r1; IL-1R1) on chromosome 1.

The floxed knock-in target I sequence (T1; interfering DNA region) was inserted between Il1r1 exon IX and exon X; it was composed of a loxP site, a mouse Engrailed-2 splice acceptor sequence (En2 SA), a destabilization domain (DD; encoding a peptide tag that would destabilize the IL-1R1 molecule), a 2xPolyA transcriptional STOP sequence, a neo cassette and a second loxP site.

The knock-in target II (T2) sequence was inserted in place of the Il1r1 stop codon in exon XI; it was composed of 3 hemagglutinin tags (3xHA), a STOP codon fluorescent protein, an internal ribosome entry site (IRES), a tdTomato fluorescent protein sequence, a single frt site and a STOP codon.

The targeting vector was electroporated into embryonic stem (ES) cells derived from a C57BL6/NTac male mouse. Correctly targeted ES cells were injected into recipient blastocysts, and chimeras were bred to C57BL/6NCrl mice to generate heterozygous mice (IL-1R1^r/+). Heterozygotes were then bred together to generate homozygous mice (IL-1R1^r/r). The IL-1R1^r colony was maintained on a C57BL/6NCrl genetic background by breeding homozygous mice together for several generations prior to sending black males to The Jackson Laboratory Repository in 2015.

Upon arrival, males were used to cryopreserve sperm. To establish the living IL-1R1^r mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).

Allele

Symbol: Il1r1^tm1Quan
Name: targeted mutation 1, Ning Quan
MGI accession ID: MGI:5633953
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Null/Knockout
Marker symbol: Il1r1
Marker name: interleukin 1 receptor, type I
Chromosome: 1
Strain of origin: C57BL/6NTac
Expressed genes: Il1r1,interleukin 1 receptor, type I,mouse, laboratory; HA,influenza hemagglutinin,; RFP,Red Fluorescent Protein,coral
Site of expression: Hemagglutinin epitope tags (HA), tdTomato, and Il1r1 will be expressed in in the cytoplasm of IL-1R1 mRNA-producing cells following Cre recombinase exposure.
Molecular note: The allele contains two knock-in sequences incorporated by two rounds of homologous recombineering. The floxed target I sequence is inserted between exon IX and exon X; it is composed of a loxP site, a mouse engrailed 2 splice acceptor sequence, a destabilization domain (DD), a 2xPolyA transcriptional STOP sequence, a neo cassette and a second loxP site. Target II sequence is inserted in place of the Il1r1 stop codon in exon XI; it is composed of 3 hemagglutinin tags (3xHA), a STOP codon fluorescent protein, an internal ribosome entry site (IRES), a tdTomato fluorescent protein sequence, a single FRT site and a STOP codon. Mice containing target I do not produce a functional protein.