NANCT Workshop 1:
Functional Approach to Identifying Transcription Factors
Laura Sichero, Aline Lopes Ribeiro, Milena Gonçalves, Cristiana Cavalieri (Instituto do Câncer do Estado de São Paulo/ICESP)
Principle
PrincipleTranscription factors (TFs) are proteins that recognize specific DNA sequences to regulatechromatin structure and gene transcription, playing a crucial role in controlling gene expression.Despite their pivotal role in various aspects of human physiology, disease, and genetic variation,understanding how TFs regulate transcription and identifying their precise binding sites remainsa significant challenge. Technological advances, such as chromatin immunoprecipitationfollowed by microarray or sequencing (ChIP-chip or ChIP-seq), have enabled the study oftranscription factor binding on a genome-wide scale. However, a critical unanswered question iswhether all binding events result in functional outcomes, underscoring the need forcomprehensive studies and functional validation, such as identifying the set of TFs that regulatespecific promoters. To address this, we constructed a comprehensive expression library of over500 human TFs and employed a luciferase reporter assay to evaluate the impact of TFsoverexpression on promoter activity. This approach enables to identify the specific set TFs orTFs complex that act on a regulatory element of interest.
Objectives
In this workshop, we will explore a series of fundamental steps for identifying transcription factors and understanding how these techniques can be applied in research. The content will be divided into the following topics:
▪ Construction of the Transcription Factors Library: We will start by explaining how a comprehensive library of transcription factors was constructed. This library is an essential tool for several studies.
▪ In Silico Analyses: Next, we will address in silico analyses using databases that help predict which TFs may interact with the regulatory region of interest. We will demonstrate how we use specific databases to make these predictions.
▪ Reporter Assay: We will then explain the reporter assay, an experimental technique used to measure the effect of TFs on the studied promoter. This assay allows us to evaluate how these factors regulate gene expression.
▪ Reverse Transfection: We will demonstrate the reverse transfection technique, where transcription factor expression vectors are co-transfected with the promoter construct, enabling us to study the effect of TFs in an in vitro model.
▪ Luminometer: To measure the results of the reporter assays, we will use the luminometer (GloMax Explorer). This step is crucial for assessing promoter activity after the overexpression of TFs.
▪ Data Interpretation: Finally, we will show how to interpret the data obtained at each step, turning raw information into meaningful conclusions. This involves analyzing results, identifying patterns, and understanding the role of the studied transcription factors.
NANCT Workshop 2:
Working with viral vectors: Biology, biosafety and practical applications
Fernanda Antunes, Bryan Strauss (Instituto do Câncer do Estado de São Paulo/ICESP)
Principle
Viral vectors continue to be used for gene transfer in laboratory and clinical applications due to their inherent ability to carry genetic material into cells. Clear examples of success in the field of gene therapy have been seen, such as the use of lentivirus for the establishment of CAR T cells (such as Kymriah and Yescarta) or the use of adenovirus for the treatment of bladder cancer (Adstiladrin). However, critical understanding of the vector’s biology, safety profile and application are necessary for its successful use. In this workshop, we will discuss the biology and biosafety of lentivirus and adenovirus. We will also have an opportunity for the participants to see how the Viral Vector Laboratory applies virus to cell cultures and assesses the phenotypic changes that result from this process.
Objectives
Provide an overview of the biology, biosafety and practical applications of lentiviral and adenoviral vectors in the study and treatment of cancer.
▪ Lentivirus: We will discuss the structure of the wild type virus and recombinant vectors, methods for production and titration, biosafety profile of the vector, and examples of the use of lentivirus for cancer gene therapy, especially CAR T cells.
▪ Adenovirus: We will discuss the structure of the wild type virus and recombinant vectors, including oncolytic adenovirus, methods for production and titration, biosafety profile of the vector, and examples of the use of adenovirus in cancer gene therapy.
Transduction: We will demonstrate the transduction processes. For this, participants will be required to use appropriate PPEs (to be provided) before entering the BSL2 facility. There we will show how the viruses are safely handled and applied to cell cultures.
▪ Detection: We will demonstrate the use of fluorescence microscopy to detect reporter genes, such as green fluorescent protein, and assays of cell death. We will also demonstrate flow cytometry assays for the detection of cell death.
NANCT Workshop 3:
Nucleic Acid Nanoparticles (NANPs) and Silver Nanoclusters (AgNCs) for Therapeutic Applications: From Design to Delivery
Kirill Afonin, Laura Rebolledo (University of North Carolina at Charlotte/UNCC)
Principle
Our lab utilizes the programmable nature of nucleic acids to develop advanced nanotechnological platforms with broad therapeutic potential. Through the development of Nucleic Acid Nanoparticles (NANPs) and DNA-templated Silver Nanoclusters (AgNCs), we aim to contribute to solutions for crucial health challenges, such as personalized drug delivery and treatments of bacterial infections. This workshop will provide some theoretical background followed by handson experience in understanding some of the approaches routinely used in the assessment of developed and optimized cutting-edge technologies and their real-world applications.