A common practice in cell biology research involves subjecting cells to a treatment regime in order to select for a subset of cells that show a particular response. It is often of great interest to understand why only a subset of cells should display the desired response. Is it due to stochastic aspects of the experiment or is it the result of intrinsic factors that predispose these cells to respond differently? What is the molecular nature of this predisposition, if any? One logical approach to address these questions is to isolate the cells that would show the desired response to the treatment, but do so in the absence of the treatment so that the cells can be studied prospectively without any untoward alterations by the treatment. Herein lies a dilemma: how to isolate cells based on their ability to respond in a certain manner to a treatment while avoiding subjecting the cells to that treatment? Here, we propose to develop an assay, termed "replica barcode selection" (RBS), that is intended to circumvent this dilemma. The assay relies on the assumption that, if a cell has a predisposition to show a certain response to a treatment due to its intrinsic genetic or epigenetic makeup, then this predisposition is likely inherited by its daughter cells after this cell divides. The RBS assay follows the following scheme: First, a unique genetic identifier, or "barcode", is inserted into each cell of the starting population. Next, these cells are allowed to proliferate such that each uniquely barcoded cell is amplified into multiple daughter cells bearing the same barcode. Cells are then split into two pools: a treatment pool and a reserve pool. The treatment pool will be subjected to the treatment to identify cells showing a positive response. Barcodes from these positive cells are then read. Finally, for each barcode identified this way, cells in the reserve pool bearing this same barcode will be retrieved. These cells should be the sisters of the positive cells in the treatment pool by virtue of having the same barcode. Cells retrieved in this manner can be studied prospectively to address whether they have a predisposition to respond to the treatment, and if so, the genetic or epigenetic basis thereof. In this grant, we propose to construct all the components of the assay and perform experiments to validate them. Once the assay is in place and has undergone initial validation, we will apply it to a proof-of-concept study of induced pluripotency. We envision broad utilities of the RBS assay in many fields of biomedical research, such as development, stem cell biology, epigenetics, cancer and aging. The successful development of the assay could therefore make significant contributions to studies of health and disease. PUBLIC HEALTH RELEVANCE: A common practice in cell biology research involves subjecting cells to a treatment regime in order to select for a subset of cells that show a particular response. Logically, one way to study why only a subset of cells - and not all the cells - show the response is to isolate these cells, but do so in the absence of the treatment so that the cells can be studied prospectively without any untoward alterations by the treatment. Herein lies a dilemma: how to isolate cells based on their ability to respond in a certain manner to a treatment regime but also avoid subjecting the cells to that treatment? Here, we propose to develop an assay, termed "replica barcode selection" (RBS), that is intended to circumvent this dilemma. We will construct all the components of the assay and perform experiments to validate them. Once the assay is in place and has undergone initial validation, we will apply it to a proof-of-concept study of induced pluripotency.