TU-Tagging: a method for studying cell type specific gene expression

Abstract

Cell type specific gene expression is a defining feature of multicellular organisms. With the advance tools such as microarrays and RNA-Seq, it is possible to study the dynamics of the gene expression during normal cellular processes as well as disease conditions. There are several methods available for cell type specific RNA isolation, the majority of which are physical cell isolation based methods, such as FACS and laser capture. However, some cell types have complex morphology such as glia and neurons, thus they have number of delicate processes, which might lose during mechanical steps of cell isolation. This RNA loss leads to lose of information and creates a need for a genetic based method, which allows RNA isolation without altering the dynamics of gene expression due to mechanical disruption.

TU-Tagging is a genetics technique which allows RNA labeling in specific cells types. It based on an enzyme called UPRT and owes its inability to distinguish uracil and uracil analog. When uracil analog (thio-uracil) provided, UPRT incorporates thio-uracil to UMP, which subsequently incorporated into RNA. Since thio-substituted nucleotides are not natural components of cell, it is possible to isolate them by biotin coupling and subsequent streptavidin isolation.

In this project, it was aimed to optimize and validate TU-Tagging by using Drosophila CNS. Basic chemistry is proven to be working while problems with cell type specificity arose and remain to be investigated.