C57BL/6-Gt(ROSA)26Sor^{tm4(CAG-Irf3*S388D*S390D)Evdr}/J

Stock number: 036261
Product name: IRF3-2D
Research areas: Diabetes and Obesity Research, Research Tools

Description

IRF3-2D mice have the 3XFlag-tagged murine Irf3 gene sequence containing two point mutations (S388D and S390D) inserted into the Gt(ROSA)26Sor locus behind a loxP-flanked STOP cassette. The IRF3-2D allele allows for tissue-specific analysis of IRF3 gain of function without the need for administration of an upstream activator such as LPS and may be useful for studying obesity and metabolism.

Detailed description

The Irf3 gene encodes an innate immune transcription factor which participates in antiviral host defense and metabolism.

The IRF3-2D knock-in allele expresses a 3XFlag-tagged murine Irf3 with two point mutations (S388D and S390D), behind a loxP-flanked STOP cassette. The IRF3-2D allele was inserted into the Gt(ROSA)26Sor locus, and therefore does not disrupt the expression of the endogenous Irf3 gene.

Prior to introduction of Cre recombinase, mice heterozygous or homozygous for the IRF3-2D allele are viable and fertile with normal breeding, and have no gross physical or behavioral abnormalities. When IRF3-2D mice are bred to mice that express Cre recombinase, the STOP cassette is removed, allowing for tissue-specific (Cre-inducible) over-expression of constitutively active IRF3 protein.
For example, when bred to adipose-specific Cre-expressing mice (Adipoq-Cre; see Stock No. 028020), the resulting mice (also called FI3OE) overexpress constitutively active IRF3 (~3-6 fold increase) in adipose tissue and show lower expression of thermogenic markers (including UCP1) in white, but not brown, adipose tissues when measured 7 days following a cold challenge. Furthermore, when fed high fat diet for 16 weeks, FI3OE mice show greater lipid accumulation in the liver, impaired insulin and glucose tolerance, and have higher fed and fasting plasma insulin levels (Yan et al., 2021).

Development

The IRF3-2D allele was created in the laboratory of Dr. Evan Rosen (Harvard Medical School) to have a 3XFlag-tagged murine Irf3 gene sequence containing two point mutations (S388D and S390D, analogous to the human S396D and S398D mutations; hereafter referred to as IRF3-2D) inserted into the Gt(ROSA)26Sor locus on chromosome 6. The specific details are below.

To generate the IRF3-2D allele, a targeting vector which contained, from 5' to 3', a chicken beta-actin promoter, a loxP-flanked stop cassette, the 3XFlag-tagged IRF3-2D sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), and a bovine growth hormone polyA signal (bGHpA). Therefore the final construct read ROSA26::CAG::loxP-STOP-loxP::3XFlag-IRF3-2D::WPRE::bGHpA. The construct was electroporated into C57BL/6N-derived ES cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with C57BL/6J for germline transmission. The resulting IRF3-2D colony was then backcrossed to C57BL/6J mice for at least 10 generations prior to sending males to The Jackson Laboratory Repository in 2021. 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).

In 2021, quality control screening performed on the mice sent to The Jackson Laboratory and the first generation rederived living colony revealed that heterozygous IRF3-2D mice genotype positive for a neomycin resistance (neo) sequence, while their wildtype littermates genotype negative for neo. This suggests the neo is closely linked to (or lies within) the IRF3-2D allele. It has not been determined if the neo is part of the stop cassette or where it lies in the IRF3-2D allele.

Allele

Symbol: Gt(ROSA)26Sor^{tm4(CAG-Irf3*S388D*S390D)Evdr}
Name: targeted mutation 4, Evan Rosen
MGI accession ID: MGI:6695703
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Epitope tag
Synonyms: IRF3-2D
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: C57BL/6N
Molecular note: The targeting vector contains from 5' to 3', a chicken beta-actin promoter, a loxP-flanked stop cassette, the 3XFlag-tagged mouse Irf3 sequence modified to carry S388D and S390D amino acid substitutions (IRF3-2D), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), and a bovine growth hormone polyA signal (bGHpA). Therefore the final construct reads ROSA26::CAG::loxP-STOP-loxP::3XFlag-IRF3-2D::WPRE::bGHpA. The S388D and S390D mutations result in a constitutively active allele without the need for an external stimulus (ie. lipopolysaccharide, poly I:C).