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In Vivo Assays for Assessing the Role of the Wilms & rsquo; Tumor Suppressor 1 (Wt1) in Angiogenesis
The Wilms & rsquo; tumor suppressor gene (WT1) is widely expressed during neovascularization, but it is almost entirely absent in quiescent adult vasculature. However, in vessels undergoing angiogenesis, WT1 is dramatically upregulated. Studies have shown Wt1 has a role in both tumor and ischemic angiogenesis, but the mechanism of Wt1 action in angiogenic tissue remains to be elucidated. Here, we describe two methods for induction of in vivo angiogenesis (subcutaneous sponge implantation, femoral artery ligation) that can be used to assess the influence of Wt1 on new blood vessel formation. Subcutaneously implanted sponges...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Methods to Identify and Validate WT1 & ndash;RNA Interaction
Tumor suppressor protein, Wt1 is a transcription factor that binds to DNA sequence similar to the Early Growth Response gene, EGR1 consensus binding sequence. Biophysical and biochemical validations have shown that the zinc fingers of Wt1 are capable of binding to both DNA and RNA albeit with different binding affinities which potentially is also isoform specific. SELEX based identification of the RNA binding motifs led to the identification of motifs which could not be translated into the in vivo context. With the advent of recent technologies that allow cross-linking of RNA and protein and high throughput sequencing tech...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

WT1-Associated Protein & ndash;Protein Interaction Networks
Tumor-suppressor protein Wt1 has been shown to interact with specific proteins that influence its function. These protein interactions have been identified as direct individual interactions but with the potential to exist as a part of a multiprotein complex. In order to obtain the global proteome interaction map of Wt1, an unbiased label-free endogenous immunoprecipitation was performed followed by mass spectrometry to identify protein interactions that are Wt1 centric. This chapter details the different techniques that have been used to identify and characterize Wt1-interacting proteins. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Function and Regulation of the Wilms & rsquo; Tumor Suppressor 1 (WT1) Gene in Fish
The Wilms & rsquo; tumor suppressor gene Wt1 is highly conserved among vertebrates. In contrast to mammals, most fish species possess two wt1 paralogs that have been named wt1a and wt1b. Concerning wt1 in fish, most work so far has been done using zebrafish, focusing on the embryonic kidney, the pronephros. In this chapter we will describe the structure and development of the pronephros as well as the role that the wt1 genes play in the embryonic zebrafish kidney. We also discuss Wt1 target genes and describe the potential function of the Wt1 proteins in the adult kidney. Finally we will summarize data on the role of Wt1 o...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Multiphoton Microscopy for Visualizing Lipids in Tissue
Visualizing the appearance of fat droplets and adipocytes in tissue can be realized using a label-free imaging method known as coherent anti-Stokes Raman spectroscopy (CARS). CARS is a nonlinear optical technique that allows label-free imaging of a material with contrast based on the same vibrational signatures of molecules found in Raman spectroscopy. CARS can be combined with other single and multiphoton imaging modes such as second harmonic generation and two-photon fluorescence to image a broad variety of biological structures. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

In Vivo Assays for Assessing the Role of the Wilms’ Tumor Suppressor 1 (Wt1) in Angiogenesis
The Wilms’ tumor suppressor gene (WT1) is widely expressed during neovascularization, but it is almost entirely absent in quiescent adult vasculature. However, in vessels undergoing angiogenesis, WT1 is dramatically upregulated. Studies have shown Wt1 has a role in both tumor and ischemic angiogenesis, but the mechanism of Wt1 action in angiogenic tissue remains to be elucidated. Here, we describe two methods for induction of in vivo angiogenesis (subcutaneous sponge implantation, femoral artery ligation) that can be used to assess the influence of Wt1 on new blood vessel formation. Subcutaneously implanted sponges s...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Isolation and Fluorescence-Activated Cell Sorting of Murine WT1-Expressing Adipocyte Precursor Cells
The current global obesity epidemic has triggered increased interest in adipose tissue biology. A major area of attention for many is adipose tissue development. A greater understanding of adipocyte ontogeny could be highly beneficial in answering questions about obesity-associated disease. Recent work has shown that a proportion of mature adipocytes in visceral white adipose tissue are derived from Wt1-expressing adipocyte precursor cells. These adipocyte precursor cells reside within the adipose tissue itself, and are a constituent of the stromal vascular fraction (SVF), along with other, non-adipogenic, cell types. Cruc...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Isolation and Colony Formation of Murine Bone and Bone Marrow Cells
Adult homeostasis is dependent on normal Wt1 expression. Loss of Wt1 expression in adult mice causes rapid loss of the mesenchymal tissues, fat and bone, amongst other phenotypes. Bone and bone marrow mesenchymal stromal cells can be studied by cell isolation and expansion. The stemness of these cells can then be characterized by carrying out a colony-forming unit-fibroblast assay and observing clonogenic capabilities. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Biological Systems and Methods for Studying WT1 in the Epicardium
The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors required for adequate heart formation. During embryonic heart formation, WT1 is mainly expressed in epicardial cells and epicardial derived cells. Its expression has been used to trace epicardial derivatives in embryos and recently it has been used to follow the reactivation of epicardial cells after myocardial infarction. Interestingly, the highest level of expression of WT1 during epicardium development correlates with the highest proliferative state, stem cell properties, and migratory capacity of epicardial cells. He...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Tools and Techniques for Wt1-Based Lineage Tracing
We describe the presently used constitutive or inducible genetic lineage tracing approaches based on the Cre/loxP system utilizing Cre recombinase expression under control of a Wt1 promoter. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

The Role of WT1 in Embryonic Development and Normal Organ Homeostasis
The Wilms’ tumor suppressor gene 1 (Wt1) is critically involved in a number of developmental processes in vertebrates, including cell differentiation, control of the epithelial/mesenchymal phenotype, proliferation, and apoptosis. Wt1 proteins act as transcriptional and post-transcriptional regulators, in mRNA splicing and in protein–protein interactions. Furthermore, Wt1 is involved in adult tissue homeostasis, kidney function, and cancer. For these reasons, Wt1 function has been extensively studied in a number of animal models to establish its spatiotemporal expression pattern and the developmental fate of the...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Clinical Aspects of WT1 and the Kidney
For more than 30 years, WT1 mutations have been associated with complex developmental syndromes involving the kidney. Acting as a transcription factor, WT1 is expressed throughout the nephron and controls the reciprocal interactions and phenotypic changes required for normal renal development. In the adult, WT1 expression remains extremely high in the renal podocyte, and at a lower level in the parietal epithelial cells. Wt1-null mice are unable to form kidneys [1]. Unsurprisingly, WT1 mutations lead to significant abnormalities of the renal and genitourinary tract, causing a number of human diseases including syndromes su...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

WT1 Mutation in Childhood Cancer
In this chapter, the role of WT1 in childhood cancer is discussed, using the key examples Wilms’ tumor, desmoplastic small round cell of childhood, and leukemia. The role of WT1 in each disease is described and mirrored to the role of WT1 in normal development. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Immunotherapy Targeting WT1: Designing a Protocol for WT1 Peptide-Based Cancer Vaccine
There is much current excitement about the potential of cancer immunotherapy. WT1 is high on the National Cancer Institute’s list of priority antigens for immune therapy. In this chapter we describe a protocol for a clinical trial using a WT1 peptide-based cancer vaccine. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news

Bioinformatic Analysis of Next-Generation Sequencing Data to Identify WT1-Associated Differential Gene and Isoform Expression
Differential gene expression analysis has been conventionally performed by microarray techniques; however with the recent advent of next-generation sequencing (NGS) approaches, it has become easier to analyze the coding as well as the noncoding components. Additionally, NGS data analysis also provides information regarding the expression changes of specific isoforms. There are several bioinformatics tools available to analyze NGS data but with different parameters. This chapter provides a comparative insight into these tools by utilizing NGS datasets available from Wt1 knockout and embryonic stem cell line model. (Source: ...
Source: Springer protocols feed by Cancer Research - December 31, 2015 Category: Cancer & Oncology Source Type: news