RNA Analysis by Biosynthetic Tagging (RABT): a tool for the identification of cell type-specific RNAs
Advancing our understanding of stem cell biology often relies on answers to the following types of questions:
What are the differences in gene expression between a stem cell and the “mature” cell (for example, a neuron or heart cell) made by the stem cell? Answers to such questions can lead to methods for directing stem cells to make specific types of progeny.
How similar are the patterns of gene expression between a “normal” cell and a stem cell-derived cell (for example, a healthy neuron in the brain versus a neuron made from an embryonic stem cell)? Answers to these types of questions can determine exactly how closely a stem cell-derived cell matches the cell it is meant to replace. This information is essential for developing safe and effective therapies.
To best answer these questions, it is necessary to study gene expression in specific cells within their normal setting. This presents a technical hurdle, since the normal setting of a cell is typically within a complex tissue, surrounded by other cell types. To perform these types of experiments using currently available tools, it is necessary to first physically remove cells of interest from all other cells. This type of manipulation can cause unwanted changes in gene expression (giving false results) and is often not technically possible.
I have developed a technique that overcomes this technical hurdle and allows the identification of genes expressed in specific cell types within a mixed population of cells. I propose to develop this technique for the study of stem cells in tissue culture and for the study of stem cells in mice. The tools that are developed as a result of this work will allow previously impossible experiments to be performed and will benefit many areas of stem cell research.
Benefits to the development of regenerative medicine therapies in California: The development of the tools described in this proposal will accelerate the progress of regenerative medicine research in California by making previously impossible experiments available to stem cell researchers. The tools described in this proposal are especially well suited for the discovery of novel biomarkers for clinically-relevant cell types, new genetic methods for directing stem cells to generate specific cell types, and new ways to functionally characterize stem cell-derived cell types; three areas of research that CIRM has identified as needing novel technologies.
Benefits to the pharmaceutical and biotechnology industry in California: The types of research that will be made possible using the proposed tools (e.g. the ability to identify cell type specific changes in gene expression in response to drug therapies or in disease states, using whole animal models) will open doors to novel areas of drug discovery, drug development, and diagnostics. Biotechnology and pharmaceutical companies will likely use these tools to launch new research efforts and ultimately new product development. Given the large presence of such industry in California, this will provide benefits to the state’s economy in the form of new jobs, new investment, and increased tax revenue.
Our project goal is to develop and test a tool that will allow the identification of stem cell-specific genes and genes that are unique to cells made by stem cells. We have made progress in applying this technique to cells grown in culture and have made the tools necessary to identify genes expressed in specific cell types using a cell culture model of nervous system development. We have also made mice that prove this technique works in genetically-manipulated mice. These mice provide important proof-of-principle results, laying the foundation for the development of additional mouse strains that can be used to identify gene expression in stem cells and other cells of interest in their normal, in vivo, setting. We have also shown that we can selectively purify the mRNAs of genes expressed in the brain and other tissues of the mouse.
Our project goal is to develop and test a tool that will allow the identification of stem cell-specific genes and genes that are unique to cells made by stem cells. We have optimized this technique in a model of nervous system development and demonstrated that we can selectively identify neural stem cell-specific RNA. We have also made progress in developing this technique in mice, with experiments demonstrating the feasibility of RNA purification from tissues known to have resident stem cell populations. Finally, we have generated mice that will allow us to identify stem cell-specific genes from normal stem cells at early stages of development and in adults as well as abnormal proliferating cells in mouse models of cancer.
Stem cells exist within complex environments, surrounded by other cells. This presents a technical challenge to researchers that want to study gene expression in stem cells without removing the cells from their natural environment. We have developed a technology that makes this possible. We used the RABT technique to purify mRNAs (gene products) from stem cells of the immune system and the nervous system, in a way that was not previously possible. We have also optimized this technique for studies in various model systems, including various tissues and developmental stages in mouse models. The tools and methods we developed should be useful to scientists in many disciplines and will allow identification of genes that regulate stem cell behavior.