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.

• Keywords
• Colon cancer, Gene targeting, Intestinal organoids, KRAS oncogene, Lung cancer, MRTX1133 inhibitor,
• MeSH
• Red Fluorescent Protein * MeSH
• DNA Nucleotidyltransferases genetics   metabolism   MeSH
• Gene Knock-In Techniques MeSH
• Luminescent Proteins * genetics   metabolism   MeSH
• Disease Models, Animal MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Lung Neoplasms genetics   pathology   MeSH
• Proto-Oncogene Proteins p21(ras) * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Red Fluorescent Protein * MeSH
• DNA Nucleotidyltransferases MeSH
• Hras protein, mouse MeSH Browser
• Luminescent Proteins * MeSH
• Proto-Oncogene Proteins 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 immunology   MeSH
• Enterobacteriaceae Infections genetics   immunology   MeSH
• Homeostasis drug effects   genetics   immunology   MeSH
• Lymphocytes drug effects   immunology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Immunity, Innate * drug effects   genetics   MeSH
• Gene Expression Regulation drug effects   MeSH
• Signal Transduction drug effects   genetics   MeSH
• Intestines drug effects   immunology   microbiology   MeSH
• Kruppel-Like Transcription Factors genetics   physiology   MeSH
• Tretinoin metabolism   pharmacology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hic1 protein, mouse MeSH Browser
• Kruppel-Like Transcription Factors 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
• Homeostasis MeSH
• Immunity MeSH
• Interleukin-17 metabolism   MeSH
• Cells, Cultured MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Gene Expression Regulation MeSH
• Repressor Proteins metabolism   MeSH
• Mucous Membrane physiology   MeSH
• Intestines physiology   MeSH
• T-Lymphocytes physiology   MeSH
• Kruppel-Like Transcription Factors genetics   metabolism   MeSH
• Tretinoin metabolism   MeSH
• Inflammation immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hic1 protein, mouse MeSH Browser
• Interleukin-17 MeSH
• Repressor Proteins MeSH
• Kruppel-Like Transcription Factors 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.

• Keywords
• Cre/loxP, TCF/LEF transcription factors, Wnt pathway, gene targeting, gut, β-catenin,
• MeSH
• beta Catenin metabolism   MeSH
• Cell Differentiation MeSH
• Cell Division MeSH
• Transcription, Genetic MeSH
• Stem Cells cytology   metabolism   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Proliferation MeSH
• Wnt Signaling Pathway * MeSH
• Intestinal Mucosa cytology   metabolism   MeSH
• Intestine, Small cytology   metabolism   MeSH
• Transcription Factor 4 MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors chemistry   genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta Catenin MeSH
• Tcf4 protein, mouse MeSH Browser
• Transcription Factor 4 MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors MeSH