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  • Protocol

    Generation of iPSC Cell Lines from Patients with Sex Chromosome Aneuploidies

    (Springer US, 2024-02-15) Astro, Veronica; Adamo, Antonio; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

    Somatic cell reprogramming allows the generation of human induced pluripotent stem cells (iPSCs) from patient's cells. The derived iPSCs provide an unlimited source of patient-specific cells that can be virtually differentiated in any cell of the human body. The generation of iPSCs has important implications for all human medicine fields, as they can be used for drug discovery, regenerative medicine, and developmental studies. Klinefelter Syndrome (KS) is the most common chromosome aneuploidy in males. KS is typically characterized by a 47,XXY karyotype, representing 80-90% of KS patients. In rare cases, high-grade sex chromosome aneuploidies (SCAs), 48,XXXY; 48,XXYY; 49,XXXXY, are also observed in males. Since the advent of the reprogramming technique, a few KS-iPSCs have been described. Here, we detail the methodology for generating primary fibroblasts from patients' skin biopsies and the subsequent derivation of iPSCs using an efficient integrative-free mRNA-based somatic reprogramming approach.

  • Protocol

    Detecting Protein-Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC) and Luciferase Complementation Assays (LCA)

    (Springer US, 2023-07-15) Bais, Pepijn; Alidrissi, Louai; Blilou, Ikram; BESE Division, Plant Cell and Developmental Biology, King Abdullah University of Science and Technology, Kingdom of Saudi Arabia, Thuwal; Plant Science; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program; Center for Desert Agriculture

    In multicellular organisms, establishing the full body plane involves cell-cell signaling where protein associations are important for the diverse cellular functions within the cells. For the study of protein-protein interactions (PPI), bimolecular fluorescence complementation (BiFC) and luciferase complementation assays (LCA) have proven to be reliable tools that can be used to confirm the physical association of two proteins in a semi-in vivo environment. This chapter provides a detailed description of these two techniques using Nicotiana benthamiana as a semi-in vivo transient expression system. As an example, we will use the interaction of the two well-described transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR), which are known as regulators of asymmetric cell division and stem cell specification in the root meristem of the model plant Arabidopsis thaliana. While the BiFC assay provides subcellular information by displaying a fluorescence signal, nuclear in this case, resulting from the reconstituted fluorophore, the LCA generates a quantitative readout of the SCR-SHR interaction. The combination of both assays provides information on the localization and strength of the PPI.

  • Protocol

    Detecting Cell Compartment-Specific PRC2-RNA Interactions via UV-RIP

    (Springer US, 2023-05-23) Della Valle, Francesco; Liu, Peng; Morelli, Gabriele; Orlando, Valerio; King Abdullah University of Science and Technology, Biological and Environmental Sciences and Engineering Division, KAUST Environmental Epigenetics Research Program, Kingdom of Saudi Arabia, Thuwal; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

    Upon cellular reprogramming, the activity of polycomb repressive complex 2 (PRC2), together with histone demethylases, is essential for the suppression of cell lineage-specific gene expression programs, for resetting of epigenetic memory and for the reacquisition of pluripotency. PRC2 requires interaction with RNAs for the correct protein complex assembly and recruitment on chromatin. Moreover, PRC2 components can be found in different cell compartments and their intracellular dynamics is part of their functional activity. Several loss-of-function studies revealed that many lncRNAs expressed upon reprogramming are essential for the silencing of lineage-specific genes and the function of chromatin modifiers. Compartment-specific UV-RIP technique is a method that will help understanding which is the nature of those interactions, with no interference from indirect interactions typical of methods involving the use of chemical cross-linkers or performed in native conditions with non-stringent buffers. This technique will shed lights on the specificity of lncRNA interaction and PRC2 stability/activity on chromatin and whether PRC2-lncRNA interaction occurs in specific cell compartments.

  • Protocol

    Dynamic Interactome of PRC2-EZH1 Complex Using Tandem-Affinity Purification and Quantitative Mass Spectrometry

    (Springer US, 2023-05-23) Liu, Peng; Zhang, Huoming; Della Valle, Francesco; Orlando, Valerio; King Abdullah University of Science and Technology, Biological and Environmental Sciences and Engineering Division, KAUST Environmental Epigenetics Research Program, Kingdom of Saudi Arabia, Thuwal; King Abdullah University of Science and Technology, Core Labs, Kingdom of Saudi Arabia, Thuwal; Proteomics and Protein Expression; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

    The Polycomb repressive complex 2 (PRC2) is a well-characterized chromatin regulator of transcription programs acting through H3K27me3 deposition. In mammals, there are two main versions of PRC2 complexes: PRC2-EZH2, which is prevalent in cycling cells, and PRC2-EZH1 where EZH1 replaces EZH2 in post-mitotic tissues. Stoichiometry of PRC2 complex is dynamically modulated during cellular differentiation and various stress conditions. Therefore, unraveling unique architecture of PRC2 complexes under specific biological context through comprehensive and quantitative characterization could provide insight into the underlying mechanistic molecular mechanism in regulation of transcription process. In this chapter, we describe an efficient method which combines tandem-affinity purification (TAP) with label-free quantitative proteomics strategy for studying PRC2-EZH1 complex architecture alterations and identifying novel protein regulators in post-mitotic C2C12 skeletal muscle cells.

  • Protocol

    Two-step protocol for extrachromosomal array integration by MosTI in C. elegans using Cas9 protein

    (2023-03-12) El Mouridi, Sonia; Frøkjær-Jensen, Christian; King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division (BESE), KAUST Environmental Epigenetics Program (KEEP), Thuwal, 23955- 6900, Saudi Arabia; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

    Transgenic Caenorhabditis elegans harboring large extrachromosomal arrays (several megabases) are relatively easy to generate, but suffer from variable and mosaic expression. Here, we describe a detailed protocol for stable transgene expression by chromosomal integration of arrays at safe-harbor landing sites or at the endogenous unc-119 locus. Our strategy is based on two rounds of injection. The first injection is a DNA-based injection to generate transgenic animals carrying extrachromosomal arrays. The second injection contains Cas9 protein and sgRNA for site-specific array integration at high frequency (44% of injected animals). Although this strategy necessitates two rounds of injections, the advantage is that no Cas9/sgRNA complex is co-integrated. Additionally, the use of an intermediate strain with a fully-formed extrachromosomal inherited array before integration favors integration of large DNA fragments.