STOCK Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy} Tg(tetO-UPF1*R844C)3438Kmke/J

Stock number: 039665
Product name: ROSA26-rtTA(Δneo); dnUPF1
Strain synonyms: dnUPF1
Research areas: Endocrine Deficiency Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Neurobiology Research, Research Tools

Description

ROSA26-rtTA(Δneo); dnUPF1 double mutant mice have the widespread rtTA-expressing ROSA26-rtTA(Δneo) allele and the dnUPF1 transgene (TRE::HA-dnUPF1-SV40pA). These double mutant mice allow tetracycline-inducible expression of a dominant-negative form of human UPF1 (hUPF1*R844C) designed to inhibit nonsense-mediated mRNA decay (NMD) in mouse tissues. These mice allow the examination of the effects of global NMD inhibition in vivo.

Detailed description

This ROSA26-rtTA(Δneo); dnUPF1 double mutant mouse line has the widespread reverse tetracycline controlled transactivator (rtTA)-expressing ROSA26-rtTA(Δneo) allele and the dnUPF1 transgene (TRE::HA-dnUPF1-SV40pA). The dnUPF1 transgene has the Tet response element/promoter (TRE; tetO) upstream of a dominant-negative form of human UPF1 (hUPF1*R844C). Once expressed, the hUPF1*R844C generates a stable form of UPF1 that can assemble into nonsense-mediated mRNA decay (NMD) complexes but lacks helicase activity, which is necessary for NMD. As such, hUPF1*R844C acts in a dominant-negative manner by reducing the number of functional NMD complexes. These ROSA26-rtTA(Δneo); dnUPF1 double mutant mice allow tetracycline-inducible examination of the effects of global NMD inhibition in vivo.

Prior to administration of the tetracycline analog doxycycline (dox), the donating laboratory reports that ROSA26-rtTA(Δneo); dnUPF1 double homozygous mice are viable and fertile with no obvious immunological or bone abnormalities.

Upon dox administration, ROSA26-rtTA(Δneo); dnUPF1 double homozygous mice exhibit robust hUPF1*R844C expression in the ileum, lung, ovary, spleen, testes, and thymus. They found mild immunological abnormalities characterized by a significant increase in total white blood cells in both male and female mice using high dox concentrations (500 mg/ml) that were not observed in low dox concentrations (125 mg/ml or lower). In addition, mild bone abnormalities including an increased trabecular number, increased connectivity density, decreased trabecular separation, and a slight decrease in cortical bone volume in female mice using high dox concentrations (500 mg/ml) - see Echols et al., 2020 Dis Model Mech [PMID:32737261]. Furthermore, they report the immunological abnormalities observed are similar to Stock No. 010939, a strain where the human beta-actin (ACTB) promoter drives constitutive, widespread expression of hUPF1*R844C.

Of note, the ROSA26-rtTA(Δneo) allele weakly expresses a downstream IRES-EGFP that is not visible using standard fluorescent microscopy.

Development

The ROSA26-rtTA(Δneo); dnUPF1 double mutant mouse line has the widespread rtTA-expressing ROSA26-rtTA(Δneo) allele and the dnUPF1 transgene. Each are described below.

The dnUPF1 transgene (TRE::HA-dnUPF1-SV40pA) was designed by the laboratory of Dr. Kim Keeling [University of Alabama at Birmingham] to contain a tetracycline response element (TRE; consisting of seven direct repeats of a 42-bp sequence containing the tet operator located just upstream of the minimal CMV promoter), a cDNA sequence encoding human UPF1 RNA helicase and ATPase (UPF1) with R844C mutation (which corresponds to R843C based on Ensenbl Canonical Transcript: ENST00000262803.10 UPF1-201 reference sequence) and N-terminal hemagglutinin (HA) epitope tag, and an SV40 transcriptional terminator (pA) sequence. The linearized construct was microinjected into C57BL/6J fertilized oocytes, which were implanted into pseudopregnant foster mothers to produce transgenic mice. Two founder lines were identified by PCR and then bred with wildtype C57BL/6 mice. The high expression line 1 with 12 to 14 copies, with an unknown insertion site, was identified and then used as follows.

The ROSA26-rtTA(Δneo) strain was generated by first obtaining Gt(ROSA)26Sor^{tm1(rtTA,EGFP)Nagy}/J mice (Stock No. 005572) and crossing it with C57BL/6-Tg(Zp3-cre)93Knw/J mice (Stock No. 003651) to excise the loxP-flanked PGK-neo-pA fragment. The resulting ROSA26-rtTA(Δneo) (Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy}) mice lacking the Zp3-cre transgene were bred in Dr. Keeling's lab to dnUPF1 mice. The donating laboratory maintained the double mutant colony by breeding as homozygous for both the ROSA26-rtTA(Δneo) allele and the dnUPF1 transgene prior to sending males 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).

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy}
Name: targeted mutation 1.1, Andras Nagy
MGI accession ID: MGI:5286599
Generation method: Targeted
Attributes: Reporter; Transactivator
Synonyms: ROSA26rtTA; Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy}
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: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Widespread reverse tetracycline transactivator (rtTA)-expression.
Molecular note: A targeting vector containing a floxed Pgk-neo-pA cassette and a rtTA-IRES-EGFP-pA cassette was inserted into intron 1 of the locus. Cre-mediated recombination removed the floxed neo cassette allowing the ROSA26 promoter to drive expression of rtTA and EGFP. Presence of doxycycline results in the formation of an active transcriptional activator and the activation of the responder transgene.

Allele

Symbol: Tg(tetO-UPF1*R844C)3438Kmke
Name: transgene insertion 3438, Kim M Keeling
MGI accession ID: MGI:7787080
Generation method: Transgenic
Attributes: Inserted expressed sequence
Synonyms: dnUPF1; UPF1-R844C
Marker symbol: Tg(tetO-UPF1*R844C)3438Kmke
Marker name: transgene insertion 3438, Kim M Keeling
Chromosome: UN
Strain of origin: C57BL/6J
Molecular note: The dnUPF1 transgene consists of a tetracycline response element (TRE) and minimal CMV promoter, hemagglutinin (HA) epitope tag fused to the N-terminus human UPF1 cDNA with an R844C mutation (which corresponds to R843C based on Ensenbl Canonical Transcript: ENST00000262803.10 UPF1-201 as a reference sequence), followed by SV40 transcriptional terminator (pA). Two funder lines were generated. This is line 3438 with 12 to 14 copies of the transgene, which shows higher and broader expression.