RNA Analysis by Biosynthetic Tagging (RABT): a tool for the identification of cell type-specific RNAs
Tools and Technologies I
Statement of Benefit to California:
The applicant aims to apply a new method known as RNA Analysis by Biosynthetic Tagging or RABT to stem cells. This technique uses cell- or tissue-specific promoters to drive expression of an RNA-modification enzyme that does not occur naturally in multicellular organisms. Cells expressing the enzyme will label their mRNA, which can then be selectively purified, obviating the need for cell purification. The principal investigator (PI) proposes to apply RABT to tissue culture cells, mouse embryonic stem cells, and enzyme-expressing transgenic mice with the goal of developing a tool for analysis of gene expression profiles in vitro and in vivo. The technique would overcome the current difficulty of identifying and isolating specific cell types among mixed cell populations and tissues. The PI intends to establish collaborations with other stem cell researchers interested in using the new RABT tools. Reviewers commented that the proposed RABT method might have a broad impact on many aspects of stem cell biology. It may well set a new standard for analysis of the stem cell transcriptome. Although several different applications are discussed, one reviewer noted that potentially the most useful will be the development of a transgenic mouse line that would allow conditional expression of the mRNA modifying enzyme in selected cell types by crossing with mouse lines expressing the Cre recombinase in specific tissues. This tool would provide a novel method for cell type-specific purification of mRNA for gene expression profiling of stem cells and their differentiated derivatives. Reviewers also felt that the feasibility of the proposed work is high based on the PI’s previous success using the method in mammalian cell lines and specific tissues within the complete Drosophila organism. The proposed project will extend the technology to the mouse model. One reviewer viewed specific aim one largely as a proof-of-principle exercise, and felt that the results may not be worth the effort required to work out conditions for introducing lenti-viral vectors. However, the reviewer felt that the second aim is more important and more likely to succeed. Overall, the proposal was judged to be well designed and feasible. The PI and co-investigator are relatively new assistant professors and they bring complimentary expertise to the project. A senior research associate and postdoctoral fellow will be recruited to the team. The PI is uniquely qualified for development of new methods based on RABT since he/she was co-inventor of this technology and has used the method in several systems since its origin. The overall strengths of the application are the potentially high impact of the tool in stem cell research, the likelihood of succeeding in the development of a useful tool, and the PI’s creativity and experience as the inventor of the new methodology. The proposal’s weakness is largely attributed to the PI’s inexperience with lenti-viral vectors and use of mouse models but reviewers viewed these as minor. Development of this tool should prove useful to stem cell scientists.