Wild type | EPFL Graph Search

The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele. "Mutant" alleles can vary to a great extent, and even become the wild type if a genetic shift occurs within the population. Continued advancements in genetic mapping technologies have created a better understanding of how mutations occur and interact with other genes to alter phenotype. It is now appreciated that most or all gene loci exist in a variety of allelic forms, which vary in frequency throughout the geographic range of a species, and that a uniform wild type does not exist. In general, however, the most prevalent allele – i.e., the one with the highest gene frequency – is the one deemed wild type. The concept of wild type is useful in some experimental organisms such as fruit flies Drosophila melanogaster, in which

Characterization of STAT1-/- mice & Investigation of IFN alpha/beta production in murine bone marrow in response to injury

Yulia Kirpicheva

The hematopoietic system is a regenerative tissue with high cell turnover. Normal homeostasis of the hematopoietic system is insured by hematopoietic stem cells (HSCs). The important features of HSCs are the capacity to self-renew and multipotency. Multipotent HSCs perpetuate through life and are able to form all differentiated cell lineages of the blood. Most HSCs with long-term self-renewal capacity are quiescent and divide infrequently. However quiescent HSCs can be activated in response to injury. The signals responsible for the activation of quiescent HSCs are poorly investigated. The Trumpp laboratory has recently identified a role for IFNα in activation of quiescent HSCs in vivo. Activation of HSCs by IFNα involves increased phosphorylation of STAT1. Therefore the role of STAT1 in the homeostasis of the hematopoietic system was investigated by characterization of the hematopoietic system in STAT1-/- mice. A 2-fold decrease in short-term HSC (ST-HSCs) and multipotent progenitors (MPPs) was observed in the bone marrow of STAT1-/- mice compared to C57Bl/6 (wild type) mice. Analysis of progenitors of HSCs also revealed a small increase in common myeloid progenitors (CMPs) and a small decrease in common lymphoid progenitors (CLPs) in the bone marrow of STAT1-/- mice. These results indicate that STAT1 play a role in the homeostasis of ST-HSCs and MPPs in bone marrow. Functional analysis of STAT1-/- LT-HSCs using in vivo long-term reconstitution assay suggests that STAT1-/- LT-HSCs can reconstitute lethally irradiated mice. Nevertheless further investigations are necessary to examine whether STAT1 plays a role in HSCs self-renewal. The unexpected role of IFNα in activation of quiescent HSCs leads to the hypothesis that endogenously produced IFNα might be important for activation of HSCs in response to injury. To address this we generated the mouse model MxCre;R26REYFP to monitor endogenous IFNα/β production in the bone marrow after injury. Our results suggest that IFNα/β is produced in response to 5-FU induced myeloid suppression and G-CSF treatment in vivo. The role of IFNα/β during the recovery phase after 5-FU induced myeloid suppression was investigated using IFNAR-/- mice. These results suggest that endogenous IFNα/β might be important for the mobilization of HSCs into the spleen induced in response to 5-FU

2008

Phase specific transcriptional regulation of circadian clock and metabolism in mouse liver

Jonathan Aryeh Sobel

The molecular clock has been conserved from cyanobacteria to mammals and is believed to align behavioral and biochemical processes with the diurnal cycle. This cellular mechanism has been an advantage to increase the fitness of organisms through the ability to anticipate food availability or predator presence. Accumulating evidence has revealed that the circadian clock is intimately interconnected with the metabolic cycle. But, the nature of these interconnections is yet not clear at the transcriptional level, and the contribution of cis-regulatory modules has not been elucidated. Accessible chromatin regions of the genome are implicated in diverse processes, such as gene regulation through enhancers and promoters, insulation of genomic domains or alternative splicing. They allow cell-type specific programs that are controlled by tissue-specific transcription factors and chromatin modifiers, although tissue-specific regulation of the circadian clock remains unclear. Therefore, we compared genome-wide BMAL1 binding and chromatin accessibility in the liver and in NIH3T3 fibroblasts. Moreover, for the first time, our study explores the dynamics of accessible regions, histone 3 lysine 27 acetylation (H3K27ac) and RNA polymerase II (Pol II) every 4 hours over one day in mouse liver. In this study, we show that a substantial fraction of these accessible sites are oscillating during a diurnal cycle with a circadian period. We observed that these accessible regions are enriched in the proximity of actively transcribed genes, and that they are dynamically affected by the binding of transcription factors such as BMAL1. We investigated wild-type (WT) and Bmal1-/- genotypes, in night restricted feeding regimen and in Light-Dark (LD) cycle, to study the circadian clock regulatory network underlying diurnal transcription. Therefore, we applied a penalized generalized linear model to infer the activity of transcription factor binding motifs in oscillating accessible sites using Pol II loadings at the transcription start sites of nearby genes. We were able to recapitulate the known regulatory elements of the circadian clock, notably E-box, D-Box, and ROR-responsive elements (RRE) in the WT genotype. On the other hand, we found that Forkhead box (FOX), glucocorticoids responsive elements (GRE), and C-AMP Response Element (CRE) were the main contributors of the regulation of oscillating genes in Bmal1-/-. Finally, using a mixture model to detect footprints with a base pair resolution, we studied the dynamics of the accessibility overlapping E-box. Our last analysis suggested that BMAL1/CLOCK is binding on double E-boxes with a spacer of 6 or 7 bp in a hetero-tetramer configuration. A 3D structure model further supported this binding mode. In Summary, we used DNase I-seq and ChIP-seq of Pol II and H3K27ac to study the circadian chromatin landscape in a 4h time-resolved experimental design. We uncovered the underlying circadian transcriptional regulatory network and, we dissected the chromatin accessibility around BMAL1 binding sites at a base pair resolution, which led to an unappreciated mode of binding of BMAL1/CLOCK in a hetero-tetramer conformation on double E-boxes. Lastly, we found tissue-specific factors that might contribute to tissue-specific binding of BMAL1/CLOCK.

EPFL2016

Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Heidi Fournier

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

2011