Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRASG12D that enables the "activation" of KRASG12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated KrasRR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the KrasRR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRASG12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRASG12D function is effectively suppressed by specific inhibitor MRTX1133.

• Klíčová slova
• Colon cancer, Gene targeting, Intestinal organoids, KRAS oncogene, Lung cancer, MRTX1133 inhibitor,
• MeSH
• červený fluorescenční protein * MeSH
• DNA-nukleotidyltransferasy genetika   metabolismus   MeSH
• genový knockin MeSH
• luminescentní proteiny * genetika   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši transgenní MeSH
• myši MeSH
• nádory plic genetika   patologie   MeSH
• protoonkogenní proteiny p21(ras) * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• červený fluorescenční protein * MeSH
• DNA-nukleotidyltransferasy MeSH
• Hras protein, mouse MeSH Prohlížeč
• luminescentní proteiny * MeSH
• protoonkogenní proteiny p21(ras) * MeSH

The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity.

• MeSH
• Citrobacter rodentium imunologie   MeSH
• enterobakteriální infekce genetika   imunologie   MeSH
• homeostáza účinky léků   genetika   imunologie   MeSH
• lymfocyty účinky léků   imunologie   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• přirozená imunita * účinky léků   genetika   MeSH
• regulace genové exprese účinky léků   MeSH
• signální transdukce účinky léků   genetika   MeSH
• střeva účinky léků   imunologie   mikrobiologie   MeSH
• transkripční faktory Krüppel-like genetika   fyziologie   MeSH
• tretinoin metabolismus   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Hic1 protein, mouse MeSH Prohlížeč
• transkripční faktory Krüppel-like MeSH
• tretinoin MeSH

The intestine is a unique immune environment that must respond to infectious organisms but remain tolerant to commensal microbes and food antigens. However, the molecular mechanisms that regulate immune cell function in the intestine remain unclear. Here we identify the POK/ZBTB family transcription factor hypermethylated in cancer 1 (HIC1, ZBTB29) as a central component of immunity and inflammation in the intestine. HIC1 is specifically expressed in immune cells in the intestinal lamina propria (LP) in the steady state and mice with a T-cell-specific deletion of HIC1 have reduced numbers of T cells in the LP. HIC1 expression is regulated by the Vitamin A metabolite retinoic acid, as mice raised on a Vitamin A-deficient diet lack HIC1-positive cells in the intestine. HIC1-deficient T cells overproduce IL-17A in vitro and in vivo, and fail to induce intestinal inflammation, identifying a critical role for HIC1 in the regulation of T-cell function in the intestinal microenvironment under both homeostatic and inflammatory conditions.

• MeSH
• homeostáza MeSH
• imunita MeSH
• interleukin-17 metabolismus   MeSH
• kultivované buňky MeSH
• myši transgenní MeSH
• myši MeSH
• regulace genové exprese MeSH
• represorové proteiny metabolismus   MeSH
• sliznice fyziologie   MeSH
• střeva fyziologie   MeSH
• T-lymfocyty fyziologie   MeSH
• transkripční faktory Krüppel-like genetika   metabolismus   MeSH
• tretinoin metabolismus   MeSH
• zánět imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Hic1 protein, mouse MeSH Prohlížeč
• interleukin-17 MeSH
• represorové proteiny MeSH
• transkripční faktory Krüppel-like MeSH
• tretinoin MeSH

The Wnt pathway plays a crucial role in self-renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N-terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild-type (wt) TCF4 protein decreased transcription of β-catenin-TCF4-responsive genes. Interestingly, suppression of Wnt/β-catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC-specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc-deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell-autonomous inhibition of β-catenin-Tcf-mediated transcription.

• Klíčová slova
• Cre/loxP, TCF/LEF transcription factors, Wnt pathway, gene targeting, gut, β-catenin,
• MeSH
• beta-katenin metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné dělení MeSH
• genetická transkripce MeSH
• kmenové buňky cytologie   metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• proliferace buněk MeSH
• signální dráha Wnt * MeSH
• střevní sliznice cytologie   metabolismus   MeSH
• tenké střevo cytologie   metabolismus   MeSH
• transkripční faktor 4 MeSH
• transkripční faktory BHLH-Zip chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• Tcf4 protein, mouse MeSH Prohlížeč
• transkripční faktor 4 MeSH
• transkripční faktory BHLH-Zip MeSH