STOCK Rai1^tm1Luo/J

Stock number: 029101
Product name: Rai1-Tag (Rai1^Tag)
Research areas: Cancer Research, Cell Biology Research, Diabetes and Obesity Research, Neurobiology Research, Research Tools

Description

The Rai1^Tag knock-in allele expresses a FLAG/myc-tagged RAI1 (Rai1-Tag) before Cre recombinase exposure. Cre-mediated deletion of the floxed FLAG-myc-STOP sequence results in expression of RAI1/EGFP fusion protein (Rai1^EGFP). These Rai1^Tag may be useful for studying Rai1 DNA binding, circuit assembly and neuronal communication in human disorders such as Smith-Magenis syndrome (SMS).

Detailed description

Haploinsufficiency of retinoic acid induced 1 (RAI1) in humans causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein with emerging functions in the expression of genes involved in circuit assembly and neuronal communication.

The Rai1^Tag knock-in allele has a loxP-flanked FLAG-myc-STOP cassette upstream of an EGFP sequence, all inserted in-frame into the endogenous stop codon of the retinoic acid induced 1 gene. Prior to Cre recombinase exposure, expression of FLAG/myc-tagged RAI1 (Rai1-Tag) is observed in a pattern consistent with the endogenous gene, and no EGFP expression is reported. Rai1-Tag is expressed in many cell types in the brain, with an onset that parallels the neuronal differentiation process and remains broadly expressed throughout the adult mouse brain - including olfactory bulb, cortex, hippocampus, striatum, thalamus, hypothalamus, cerebellum and brainstem.


When bred to mice that express Cre recombinase, offspring will have the floxed region (FLAG-myc-STOP) deleted in cre-expressing tissues; resulting in expression of an RAI1/EGFP fusion protein (Rai1^EGFP) instead of Rai1-Tag. For example, when bred to mice with Cre-expression in subcortical excitatory neurons (Vglut2-ires-cre ; Stock No. 016963), the resulting Vglut2^Cre;Rai1^Tag mice have Cre-positive thalamic neurons expressing RAI1/EGFP but not Tag. Any Cre-negative neurons in those mice express Rai1-Tag but not EGFP.

Mice homozygous for the Rai1^Tag knock-in allele are viable and fertile with no reported gross physical or behavioral abnormalities. The Tag peptide on the Rai1 C-terminus does not adversely affect Rai1-Tag expression, and allows for immunoprecipitation (via anti-FLAG antibodies). Expression of the RAI1/EGFP fusion protein is detectable by antibody staining. However, the donating investigator reports that detection of the fusion protein by direct fluorescence has not been successful to date (December 2016).

Development

The Rai1^Tag knock-in allele was created by Dr. Liqun Luo (Stanford University) to have a loxP-flanked FLAG-myc-STOP cassette upstream of an EGFP sequence, all inserted in-frame into the endogenous stop codon of the retinoic acid induced 1 gene (Rai1) on chromosome 11. Specific details below.


First, a targeting vector was designed with (from 5' to 3') a loxP site, 3 copies of a FLAG peptide sequence (3xFLAG), 5 copies of a myc peptide sequence (5xMyc), a triple translational stop codon array (3xSTOP), a frt-flanked neo cassette (frt-pSV40-Neo-pA-frt). a second loxP site and an enhanced green fluorescent protein gene (EGFP).

The targeting vector (loxP::3xFLAG-5xMyc-3xSTOP::frt::pSV40-neo-pA::frt::loxP::EGFP) was electroporated into 129S1/Sv-derived embryonic stem (ES) cells.

Chimeras were mated with C57BL/6J wildtype mice for germline transmission.

The resulting Rai1^Tag mice were maintained on a mixed genetic background (CD1;C57BL/6J;129S1) for several generations prior to sending males with white coat color to The Jackson Laboratory Repository in 2017.

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).

Allele

Symbol: Rai1^tm1Luo
Name: targeted mutation 1, Liqun Luo
MGI accession ID: MGI:5816453
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Rai1
Marker name: retinoic acid induced 1
Chromosome: 11
Strain of origin: 129S1/Sv
Expressed genes: GFP,Green Fluorescent Protein,; FLAG, FLAG octapeptide,
Site of expression: Prior to Cre recombinase exposure, FLAG/myc-tagged RAI1, consistent with the endogenous gene, is expressed in many cell types in the brain. When bred to mice that express Cre recombinase, EGFP is expressed in in cre-expressing tissues of the offspring.
Molecular note: The targeting vector contains a loxP-flanked FLAG-myc-STOP cassette upstream of an EGFP sequence, all inserted in-frame into the endogenous stop codon. Prior to Cre recombinase exposure, expression of FLAG/myc-tagged RAI1 is observed in a pattern consistent with the endogenous gene, and no EGFP expression is reported.