FVB-Tg(KRT14-MAP2K1/Esr1)12Pkha/J

Stock number: 006822
Product name: Mek1:ER
Research areas: Cancer Research, Cell Biology Research, Dermatology Research, Research Tools

Description

The transgene in these mice contains a human keratin 14 promoter driving the expression of a fusion protein consisting of a constitutively active mutant human mitogen activated protein kinase-kinase 1 joined to the Gly525Arg mutant ligand binding domain of the mouse estrogen receptor. The fusion protein's activity is inducible by 4-hydroxytamoxifen, but unresponsive to natural estrogen.

Detailed description

Mice hemizygous for this "K14-Mek1:ER" transgene are viable and fertile, with expression of the Mek1:ER fusion protein (a constitutively active mutant region from human mitogen activated protein kinase-kinase 1 (Mek1^R4F; containing an amino-terminal deletion of aa 32-51 and the S218E/S222D substitutions) fused at its carboxy terminal with the G525R mutant murine estrogen receptor ligand binding domain (ER^TM)) directed to epidermis by the human keratin 14 promoter. Because of the ER^TM region of the fusion protein, Mek1:ER is restricted to the cytoplasm and the biochemical activity of the Mek1:ER fusion gene can be induced following tamoxifen administration. For example, induction of this constitutively active form of human Map2k1 (Mek1^R4F) promotes the undifferentiated, proliferative phenotypic characteristics observed in epidermal neoplasia in as few as 5 days (including hyperplasia, increased levels of phosphorylated ERK1/2, increased mitotic index, decreased expression of differentiation markers and increased expression of beta-1 and beta-4 integrin subunits). Mek1^R4F-induced skin abnormalities were entirely reversed within ten days after 4OHT administration cessation. These mice have a similar 4OHT-inducible skin phenotype as the transgenic mice expressing human H-Ras^G12V (Stock No. 006403) or human Raf-1[DD] (Stock No. 006661), and may be useful in studies of the Ras/Raf/MEK/ERK cell proliferation and differentiation pathway, as well as in elucidating cellular events preceding skin cancer cell transformation.

The G525R mutant murine estrogen receptor ligand binding domain (ER^TM) does not bind its natural ligand (17β-estradiol) at physiological concentrations; but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, ER^TM fusion proteins can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

The “K14-Mek1:ER” transgene was designed with a 2075-bp human keratin 14 promoter sequence upstream of the constitutively active mutant region from human mitogen activated protein kinase-kinase 1 (Mek1^R4F; containing an amino-terminal deletion of aa 32-51 and the S218E/S222D substitutions) fused at its carboxy terminal with the G525R mutant murine estrogen receptor ligand binding domain (ER^TM), which renders transgene expression tamoxifen (4OHT) inducible yet estrogen insensitive. This transgene was injected into the pronuclei of FVB embryos and chimeric founders were established. Mice from founder line 12 were then backcrossed to FVB inbred mice for at least 8 generations prior to arrival at The Jackson Laboratory.

Allele

Symbol: Tg(KRT14-MAP2K1/Esr1)12Pkha
Name: transgene insertion 12, Paul A Khavari
MGI accession ID: MGI:3700387
Generation method: Transgenic
Attributes: Inducible; Constitutively active; Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(KRT14-MAP2K1/Esr1)12Pkha
Marker name: transgene insertion 12, Paul A Khavari
Chromosome: UN
Strain of origin: FVB
Expressed genes: MAP2K1,mitogen-activated protein kinase kinase 1,human
Molecular note: The transgene contains a 2075-bp human keratin 14 promoter construct with a 5' intron from a beta-globin gene driving expression of a cDNA that encodes a constitutively active mutant human mitogen activated protein kinase 1 (with deletion of amino acids 32-51, which comprise a putative alpha-helical domain, and the amino acid substitutions Ser218Glu and Ser222Asp, which remove phosphorylatively regulable serines). This is joined at its COOH-terminal with the Gly525Arg mutant ligand-binding domain of the mouse estrogen receptor (ER^TM), which renders the fusion protein's activity inducible by 4-hydroxytamoxifen, but unresponsive to natural estrogen (17beta-estradiol).
General note: Of three PCR-confirmed transgenic mouse lines, two - designated lines 12 and 13 - express the fusion protein in skin. Topical tamoxifen treatment induces similar skin phenotypes in both lines that are more severe in line 12, which was therefore used in further studies.