In these transgenic mice, tamoxifen inducible expression of the human H-ras protein fused with the G525R mutant murine estrogen receptor ligand binding domain, is directed to epidermis by the human keratin 14 promoter.
Paul Khavari, Stanford University
Mice hemizygous for this "K14-ER:Ras" transgene are viable and fertile, with expression of the ER:Ras fusion protein (constitutively active G12V mutant form of the catalytic domain of human H-ras (H-rasV12) fused at its amino terminal with the G525R mutant murine estrogen receptor ligand binding domain (ERTM)) directed to epidermis by the human keratin 14 promoter. Because of the ERTM region of the fusion protein, ER:Ras is restricted to the cytoplasm and the biochemical activity of the ER:Ras fusion gene can be induced following tamoxifen administration. For example, prolonged (4 weeks) induction of human H-RasG12V 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. H-RasG12V-induced skin abnormalities were entirely reversed within one month after 4OHT cessation. These mice have a similar 4OHT-inducible skin phenotype as the transgenic mice expressing human Raf-1[DD] (Stock No. 006661) or human Mek1R4F (Stock No. 006822), and may be useful in studies of the Ras/Raf/MEK/ERK cell proliferation and differentiation pathway, as well as to elucidate cellular events preceding skin cancer cell transformation.
The G525R mutant murine estrogen receptor ligand binding domain (ERTM) 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, ERTM 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.
The K14-ER:Ras transgene was designed with a 2075-bp human keratin 14 promoter sequence upstream of the constitutively active G12V mutant form of the catalytic domain of human H-ras (H-rasV12) fused at its amino terminal with the G525R mutant murine estrogen receptor ligand binding domain (ERTM), which renders transgene expression tamoxifen (4OHT) inducible yet estrogen insensitive. 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.
|Expressed Gene||HRAS, Harvey rat sarcoma viral oncogene homolog, human|
|Site of Expression|
|Allele Name||transgene insertion 1, Paul A Khavari|
|Allele Type||Transgenic (Constitutively active, Humanized sequence, Inducible, Inserted expressed sequence)|
|Gene Symbol and Name||Tg(KRT14-Esr1/HRAS)1Pkha, transgene insertion 1, Paul A Khavari|
|Promoter||KRT14, keratin 14, type I, human|
|Expressed Gene||HRAS, Harvey rat sarcoma viral oncogene homolog, human|
|Strain of Origin||C57BL/6|
|General Note||Multiple mouse lines were generated, with varying transgene copy numbers and expression levels of the fusion protein. Lines were identified only as H, M, and L for high, medium and low copy number, respectively.|
|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 HRAS, in which glycine at amino acid position12 is replaced by arginine (G12V), joined at its amino terminal to a mutant mouse estrogen receptor ligand-binding domain in which replacement of glycine by arginine at amino acid position 525 (G525R, "ERTM") renders the fusion protein's activity inducible by 4-hydroxytamoxifen, but unresponsive to natural estrogen (17beta-estradiol).|
|Mutations Made By|| |
Paul Khavari, Stanford University
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