Figure 2
From: Ductal activation of oncogenic KRAS alone induces sarcomatoid phenotype
Elastase I-driven oncogenic KRASG12V expression induces SDC-like tumors in the SMGs.
(A) The diagram of the Ela-CreERT and LGL-KRasG12V transgenes26,30. Expression of the TAM-inducible Cre recombinase (CreERT) is driven by elastase I promoter/enhancer. KRASG12V expression is blocked by a loxP-GFP-Stop-loxP cassette (LGL). A mouse BAC of 222-kb carrying the intact elastase I gene was used to provide the endogenous native context of elastase I gene26. CreERT removes STOP cassette (LGL) through recombination, allowing the KRASG12V expression. (B) Elastase I-driven recombination in mTmG and Rosa26R reporter mice ascertains ductal original. Green fluorescence was exclusively detected in the ductal cells of mTmG;Ela-CreERT reporter mice after vehicle (panel a) or TAM-feeding (panel b). Scale bar: 50 μm (panel a). β-galactosidase activity was observed in the SMG ductal cells of Rosa26R;Ela-CreERT mice after TAM-feeding (panel d) compared to control mice (panel c). Scale bar: 200 μm. (C) Characterization of SMGs of LGL-KRasG12V;Ela-CreERT mice on day-24 post TAM-feeding. Gross anatomy revealed large ventrolateral cervical masses in SMGs of LGL-KRasG12V;Ela-CreERT mice on day-24 (panel b). H&E and immunohistochemical (IHC) staining showed the microscopic abnormalities in the SMGs of TAM-fed LGL-KRasG12V;Ela-CreERT mice on day-24 (panels d, f, h). Scale bars: 800 μm (in panels c and d); 100 μm (panels e–h). (D) Wet weight of SMG from LGL-KRasG12V;Ela-CreERT mice at 15-days post TAM-feeding (n = 13) or not (n = 7). ***p < 0.001. (E) Overall survival of mice after KRASG12V induction is drastically reduced. Percent survival of Ela-CreERT versus LGL-KRasG12V;Ela-CreERT mice after TAM administration. Median survival of LGL-KRasG12V;Ela-CreERT mice was 28-days. n = 13 per group; p < 0.0001 by Log-rank (Mantel-Cox) test. (F) KRASG12V is activated in the SMG tumors of LGL-KRasG12V;Ela-CreERT mice following TAM-gavage. Transgenic mice of indicated genotypes were gavaged with TAM. SMG samples were harvested on day-24 and whole tissue lysates (1 mg) from each sample were pulled down with Raf-1 RBD agarose beads. Pulldown reactions were resolved by SDS-PAGE (12%) and Western blotting was performed with an anti-KRAS antibody to detect active (GTP-bound) KRASG12V.
