Ewing Sarcoma-Specific (Re)Expression Models

Gene expression and knockdown systems are powerful tools for studying the function of single genes and their interactions within pathways. Plasmid transfection and viral transduction have revolutionized the field of molecular biology and paved the way for various gene-editing strategies such as TALEN, Zink finger nucleases, and ultimately CRISPR. In Ewing sarcoma (EwS), almost as many genes are repressed by the expression of EWSR1-FLI1 as are upregulated by the fusion oncogene. Here, we present a useful point-by-point protocol for generating transgene expression systems in EwS that allow (conditional) re-expression of a gene of interest. We provide an extensive instruction on molecular cloning, plasmid generation, viral transduction, and expression validation. Finally, we address common problems and highlight potential pitfalls that can be easily avoided with thoughtful guidance.