B6;129S4-Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe} Col1a1^{tm1(tetO-GFP,-PRKAA1*)Rjsh}/J

Stock number: 034797
Product name: iAMPK^CA
Research areas: Diabetes and Obesity Research, Research Tools

Description

iAMPK^CA mice allow Tet-On expression of a constitutively active human protein kinase, AMP-activated, alpha 1 catalytic subunit (PRKAA1 or AMPKα1) and an EGFP reporter. These mice may be useful for studying metabolic disease, including nonalcoholic fatty liver disease (NAFLD).

Detailed description

PRKAA1 (protein kinase, AMP-activated, alpha 1 catalytic subunit or AMPKα1) encodes an AMP-activated protein kinase that functions as a master regulator of metabolism. Activation of PRKAA1 (AMPKα1) restores energy balance by inhibiting ATP consumption and promoting ATP generation and is associated with an improved outcome for several metabolic diseases including type 2 diabetes and obesity.

Stock No. 034797 has the iAMPK^CA allele (Col1a1^{tm1(tetO-GFP,-PRKAA1*)Rshaw}) as well as the Rosa26-CAGs-LSL-RIK knock-in allele (RLR; Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe}). Mice homozygous for both the iAMPK^CA and the RLR allele are viable and fertile.

The iAMPK^CA allele has a Tet-responsive element (TRE or tetO) directing expression of a constitutively active truncated human PRKAA1 inserted into an intergenic site downstream of the Col1a1 locus. The truncated PRKAA1 protein contains amino acids 1-312, which includes the kinase domain, and exhibits high activity. The construct is inserted downstream of Col1a1 and does not affect the expression of any endogenous genes.

The Rosa26-CAGs-LSL-RIK (or RLR) knock-in allele has a CAG promoter and loxP-flanked STOP cassette upstream of the third-generation reverse tetracycline-regulated transactivator gene (rtTA3), IRES sequence and the monomeric far-red fluorescent protein mKate2, all inserted into the Gt(ROSA)26Sor locus. In the absence of Cre recombinase, a floxed STOP cassette prevents expression of rtTA3 and mKate2. Following cre-mediated deletion of the floxed-STOP cassette, mKate2 fluorescence is observed - and subsequent expression of a target gene under control of a tetracycline-responsive promoter element (TRE; or tetO) can be induced/regulated by tetracycline or its analog doxycycline (dox).

When iAMPK^CA ; RLR mice are combined with Tg(Alb-cre)21Mgn mice having Cre recombinase expression in the liver (Stock No. 003574), AMPK activation in progeny exposed to tetracycline or its analog doxycycline (dox) develop protection against weight gain, liver steatosis and inflammation on a high fat diet.

Development

The targeting construct includes a Tet-responsive element (TRE or tetO) directing expression of an eGFP reporter followed by an IRES and a truncated (amino acids 1-312) human protein kinase, AMP-activated, alpha 1 catalytic subunit (PRKAA1 or AMPKα1) cDNA. The construct, a "Flp-In" promoter cassette (PGK-promoter-ATG-FRT) and a pCAGGS-FlpE plasmid were electroporated into modified (C57BL/6 x 129S4/SvJae)F1-derived D34 embryonic stem (ES) cells that had previously been targeted with a Col1a1 "Flp-In" integration cassette (Col1a1-FRT-hygro-pA) and Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe} (Stock No. 029633). Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to C57BL/6J mice and progeny crossed to C57BL/6J for at least six generations by the donating laboratory (see SNP note below). Upon arrival, mice were bred to C57BL/6J for at least one generation to establish the colony.

In 2021, 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 rederived living colony at The Jackson Laboratory Repository. Four of the 43 markers throughout the genome were segregating with 129, suggesting an incomplete backcross.

Allele

Symbol: Col1a1^{tm1(tetO-GFP,-PRKAA1*)Rjsh}
Name: targeted mutation 1, Reuben J Shaw
MGI accession ID: MGI:6472613
Generation method: Targeted
Attributes: Reporter; Inducible; Inserted expressed sequence
Synonyms: iAMPK^CA; Col1a1^{tm1(tetO-GFP,-PRKAA1*)Rshaw}
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Marker synonyms: CAFYD; Col1a-1; Cola1; Cola-1; COLIA1; EDSARTH1; EDSC; Mov-13; OI1; OI2; OI3; OI4
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: The targeting construct includes a Tet-responsive element (TRE or tetO) directing expression of an eGFP reporter followed by an IRES and a truncated (amino acids 1-312) human protein kinase, AMP-activated, alpha 1 catalytic subunit (PRKAA1 or AMPKalpha;1) cDNA. The construct, a "Flp-In" promoter cassette (PGK-promoter-ATG-FRT) and a pCAGGS-FlpE plasmid were electroporated into modified (C57BL/6 x 129S4/SvJae)F1-derived D34 embryonic stem (ES) cells.
General note: The embryonic stem cell line used is (C57BL/6 x 129S4/SvJae)F1-derived D34 that had previously been targeted with a Col1a1 "Flp-In" integration cassette (Col1a1-FRT-hygro-pA) and Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe}.

Allele

Symbol: Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe}
Name: targeted mutation 2, Scott Lowe
MGI accession ID: MGI:5638918
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Transactivator
Synonyms: R26Sor^{tm2(CAGs-LSL-RIK)Slo}
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/6J x 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: When these mutant mice are bred to mice that express Cre recombinase, the resulting offspring will express rtTA3 in cre-expressing tissues with subsequent expression of RFP (mKate2) under control of a tetracycline-responsive promoter element (TRE; or tetO) that can be induced/regulated by tetracycline or its analog doxycycline (dox).
Molecular note: The targeting construct introduced into the locus a CAG promoter, floxed stop cassette, reverse tetracycline transactivator 3 (rtTA3), IRES and a far red reporter gene (mKate2).
General note: ES cell line = C10, not C2 (Jaenisch) as reported by original reference. C10 ES cells carry Col1a1.