129S.B6-Tg(KRT14-RAF1/ESR1)1Pkha/J

Stock number: 006661
Product name: K14-Raf:ER
Research areas: Cancer Research, Cell Biology Research, Dermatology Research, Research Tools

Description

Hemizygous mice express the Raf:ER fusion protein (human Raf-1 fused with the G525R mutant human estrogen receptor ligand binding domain), directed to the epidermis by the human keratin 14 promoter. Biochemical activity of the Raf:ER fusion gene, restricted to the cytoplasm , can be induced following tamoxifen administration.

Detailed description

Mice hemizygous for this "K14-Raf:ER" transgene are viable and fertile, with expression of the Raf:ER fusion protein (a constitutively active Y340D/Y341D mutant form of the catalytic domain of human Raf-1 (Raf-1[DD]) fused at its carboxy terminal with the G525R mutant human 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, Raf:ER is restricted to the cytoplasm and the biochemical activity of the Raf:ER fusion gene can be induced following tamoxifen administration. For example, prolonged (4 weeks) induction of human Raf-1[DD] activity promotes the undifferentiated, proliferative phenotypic characteristics observed in epidermal cancer; including hyperplasia, increased mitotic index, decreased expression of differentiation markers and increased expression of beta-1 and beta-4 integrin subunits. Raf-1[DD]-induced skin abnormalities are entirely reversed within one month after 4OHT cessation. These mice have a similar 4OHT-inducible skin phenotype as the transgenic mice expressing human H-Ras^G12V (Stock No. 006403) or human Mek1^R4F (Stock No. 006822), 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 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-Raf:ER” transgene was designed with a 2075-bp human keratin 14 promoter sequence upstream of the constitutively active Y340D/Y341D mutant form of the catalytic domain of human Raf-1 (Raf-1[DD]) fused at its carboxy terminal with the hormone binding domain of the human estrogen receptor. This transgene was injected into the pronuclei of C57BL/6 embryos and chimeric founders were established. Mice were then backcrossed to “129/SvEv” mice for 5 generations prior to arrival at The Jackson Laboratory.

Allele

Symbol: Tg(KRT14-RAF1/ESR1)1Pkha
Name: transgene insertion 1, Paul A Khavari
MGI accession ID: MGI:3700057
Generation method: Transgenic
Attributes: Inducible; Constitutively active; Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(KRT14-RAF1/ESR1)1Pkha
Marker name: transgene insertion 1, Paul A Khavari
Chromosome: UN
Strain of origin: C57BL/6
Expressed genes: RAF1,Raf-1 proto-oncogene, serine/threonine kinase,human; ESR1,estrogen receptor 1,human
Molecular note: The transgene contains a 2075-bp human keratin 14 promoter construct driving expression of a cDNA that encodes a fusion protein comprising a constitutively active mutant form of the catalytic domain of human RAF1, in which tyrosine is replaced by aspartic acid at amino acid positions 340 and 341 (Y340D/Y341D), joined at its carboxyl-terminal to the ligand-binding domain of human estrogen receptor 1. The fusion protein can be activated in the epidermis by topical application of 4-hydroxytamoxifen.
General note: Three independent transgenic mouse lines were generated that exhibited similar levels of fusion protein expression.