Comparison of Prox1 expressing and Prox1 knockout intestinal tumors and the effect of A2V+CD40
ABSTRACT: Control or A2V+CD40 treated Prox1-proficient (AP) and Prox1-deficient (APP) tumors Overall design: Tumors induced in Apcflox/flox;Tp53flox/flox;villin-CreERT2 (AP) or Apcflox/flox;Tp53flox/flox;Prox1flox/flox;villin-CreERT2 (APP) mice for two weeks. Mice were randomized and treated with PBS, control IgG, or A2V+CD40 for two weeks.
Project description:We isolated and selected intestinal adenoma organoids from villin-CreER; Apcflox/flox and villin-CreER; Apcflox/flox; Prox1flox/flox mice and added tamoxifen to induce the deletion of the Apc and Prox1 genes in the intestinal epitheliul ex vivo. Microarray experiments were carried out 7 days after the addition of tamoxifen. Total RNA obtained from villin-CreER; Apcflox/flox and villin-CreER; Apcflox/flox; Prox1flox/flox organoids were compared 7 days after the addition of tamoxifen and 5 days after the selection for Apc-mutant organoids in the absence of the Wnt-agonist R-Spondin1.
Project description:We made intestinal organoid cultures from Villin-CreERT2;Apcflox/flox mice. Organoids were subjected to 4-OH-Tam treatment, whereafter Apc mutant organoids were selected in R-Spondin free medium. DBZ or DMSO treatment was started on day 3. On day 8, organoids were dissociated to obtain single cells and analyzed using the Chromium Single-cell 3'RNA-sequencing system Overall design: We analyzed in total 2 samples (1 DBZ and 1 DMSO), both samples contained 3000 cells
Project description:Mutations in TGFBR2, a component of the transforming growth factor (TGF)-? signaling pathway, occur in high-frequency microsatellite instability (MSI-H) colorectal cancer (CRC). In mouse models, Tgfbr2 inactivation in the intestinal epithelium accelerates the development of malignant intestinal tumors in combination with disruption of the Wnt-?-catenin pathway. However, no studies have further identified the genes influenced by TGFBR2 inactivation following disruption of the Wnt-?-catenin pathway. We previously described CDX2P-G19Cre;Apcflox/flox mice, which is stochastically null for Apc in the colon epithelium. In this study, we generated CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice, with simultaneous loss of Apc and Tgfbr2. These mice developed tumors, including adenocarcinoma in the proximal colon. We compared gene expression profiles between tumors of the two types of mice using microarray analysis. Our results showed that the expression of the murine homolog of GSDMC was significantly upregulated by 9.25-fold in tumors of CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice compared with those of CDX2P-G19Cre;Apcflox/flox mice. We then investigated the role of GSDMC in regulating CRC tumorigenesis. The silencing of GSDMC led to a significant reduction in the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GSDMC enhanced cell proliferation. These results suggested that GSDMC functioned as an oncogene, promoting cell proliferation in colorectal carcinogenesis. In conclusion, combined inactivation of both Apc and Tgfbr2 in the colon epithelium of a CRC mouse model promoted development of adenocarcinoma in the proximal colon. Moreover, GSDMC was upregulated by TGFBR2 mutation in CRC and promoted tumor cell proliferation in CRC carcinogenesis, suggesting that GSDMC may be a promising therapeutic target.
Project description:We isolated and selected intestinal adenoma organoids from Apcmin/+; Rosa26LSL-TdTomato; Prox1-CreERT2 mice. After the selection procedure without growth factors, we induced CreERT2 activity and the transcription of tdTomato to label Prox1+ cells by 300 nM 4-hydroxytamoxifen for 16h. tdTomato+ (Prox1+) and tdTomato- cells (enriched for Prox1- cells) were FACS sorted and total RNA was isolated. Overall design: Total RNA obtained from tdTomato+ (Prox1+) cells was compared to the tdTomato- cell population (enriched for Prox1- cells) after FACS sorting.
Project description:Intestinal epithelial cells (IECs) were isolated from the colon of Villin-CreERT2, Rnf20-flox and Rnf40-flox mice two weeks upon the Tamoxifen-induced, intestinal knockout of Rnf20 and Rnf40. RNA was isolated from snap-frozen IECs to perform mRNA-seq.
Project description:Intestinal epithelial cells (IECs) were isolated from the colon of Villin-CreERT2, Rnf20-flox and Rnf40-flox mice two weeks upon the Tamoxifen-induced, intestinal knockout of Rnf20 and Rnf40. ChIP-seq for H3K4me3 was performed using snap-frozen IECs.
Project description:Intestinal crypts isolated from Apcflox/flox; villin-CreERT mice were treated with Tamoxifen to induce the deletion of Apc. Tamoxifen-treated organoids were selected in the absence of Wnt agonists and then treated with TGF-beta. Total RNA obtained from Tamoxifen-treated, Apc-deleted intestinal organoids in the absence or presence of 3 ng/ml TGF-beta (18h).
Project description:GATA6 is a transcription factor involved in the differentiation of intestinal epithelial cells into differentiated absorptive epithelial cells. GATA6 is expressed in all segments of the small intestine. We examined the impact of deleting GATA6 from intestinal epithelial cells of the adult ileum 6-8 week old Villin-CreERT2 mice or Villin-CreERT2 mice crossed to GATA6 flox/flox mice were treated with a single injection of tamoxifen. (100 mg/ml) for 5 consecutive days. 28 days later the ileum was dissected and RNA prepared from the whole ileal segment using the RNAeasy method with DNAse treatment. Three individual mice were examined in each of the control and GATA6 KO groups.
Project description:With the tamoxifen-inducible CreERT2 system, genetic recombination can be temporally controlled in a cell-type-specific manner in intact animals, permitting dissection of the molecular underpinnings of mammalian physiology. Here we present a significant drawback to CreERT2 technology for analysis of intestinal stem cells. Using the intestine-specific Villin-CreERT2 mouse strain, we observed delayed intestinal regeneration post irradiation. Villin-CreERT2 activation was associated with DNA damage and cryptic loxP site cleavage. Analysis of stem cell-specific CreERT2 strains showed that the genome toxicity impairs function of crypt base columnar stem cells, resulting in loss of organoid initiating activity. Importantly, the stem cell impairment is short-lived, with return to normal by 7 days post tamoxifen treatment. Our findings demonstrate that mouse genetic experiments that utilize CreERT2 should consider the confounding effects of enhanced stem cell sensitivity to genome toxicity resulting from CreERT2 activation.
Project description:We carried out an analysis of the expression of Prox1, a homeo-domain transcription factor, during mouse inner ear development with particular emphasis on the auditory system. Prox1 is expressed in the otocyst beginning at embryonic day (E)11, in the developing vestibular sensory patches. Expression is down regulated in maturing (myosin VIIA immunoreactive) vestibular hair cells and subsequently in the underlying support cell layer by E16.5. In the auditory sensory epithelium, Prox1 is initially expressed at embryonic day 14.5 in a narrow stripe of cells at the base of the cochlea. This stripe encompasses the full thickness of the sensory epithelium, including developing hair cells and support cells. Over the next several days the stripe of expression extends to the apex, and as the sensory epithelium differentiates Prox1 becomes restricted to a subset of support cells. Double labeling for Prox1 and cell-type-specific markers revealed that the outer hair cells transiently express Prox1. After E18, Prox1 protein is no longer detectable in hair cells, but it continues to be expressed in support cells for the rest of embryogenesis and into the second postnatal week. During this time, Prox1 is not expressed in all support cell types in the organ of Corti, but is restricted to developing Deiters' and pillar cells. The expression is maintained in these cells into the second week of postnatal life, at which time Prox1 is dynamically down regulated. These studies form a baseline from which we can analyze the role of Prox1 in vertebrate sensory development.