Transgenic ES Cell Differentiation Systems to replace Transgenic Mouse Analysis of Tissue Specific Regulatory Elements

Understanding how genes are regulated has important implications in both normal development and in the progression of diseases such as cancer. For a specific cell or tissue to develop, the appropriate set of genes must be activated in a tightly controlled process. Regulatory elements that control overall gene activation are currently identified using a combination of cell based assays and transgenic mice to assess the cell types in which they are active. A major drawback of analysing regulatory elements in this way are the animal welfare issues concerning both the procedures that the mice undergo as well as the numbers of mice required.
Here, we are proposing to develop reagents and protocols that will replace the use of transgenic mice with in vitro based embryonic stem (ES) cell differentiation assays. ES cells readily undergo genetic manipulation and under appropriate culture conditions have the potential to develop into many different cell types. We plan to systematically test regulatory elements during ES cell differentiation and validate the cell types that can be studied in this way in order to demonstrate that ES cell differentiation provides a viable alternative for assessing gene regulatory function. By providing suitable tools and protocols we aim to encourage the wider scientific community to adopt ES cell based systems for analysing gene regulation thereby dramatically reducing the numbers of animals used in this kind of research. In addition, this study will help to identify regulatory elements that are specifically active in various cell types with potential therapeutic benefits such as immune cells or insulin producing pancreatic cells. ES cells engineered to carry such cell-type specific regulatory elements will provide powerful tools for the development of new protocols for producing these cells and will therefore be vital to harness the promise of embryonic stem cells for regenerative medicine.

Identifying the cis-regulatory mechanisms controlling gene expression will be crucial to understand not only how normal cellular development proceeds but also how dysregulation of transcription causes disease. At present, the definitive assay for attributing tissue specific function to regulatory elements is the generation of transgenic reporter mice resulting in significant use of animals (e.g. 15% of the total number of animals purchased by CBS, the core animal research facility for the Addenbrooke?s biomedical research campus). Development of alternative non-animal assays therefore offers very significant scope for reducing animal use across the UK and beyond.
Here, we propose to develop reagents and protocols that will lead to a substantial replacement in the use of transgenic mice for analysing regulatory elements with in vitro based transgenic embryonic stem (ES) cell differentiation assays. Building on current ES cell targeting strategies we aim to modify and develop a toolbox of vectors that permits regulatory elements to be easily integrated into a single site in the genome. By robustly testing the activity of regulatory elements during the in vitro differentiation of ES cells and validating the cell types that can be targeted we intend to demonstrate that ES cell based assays provide an exciting and viable alternative to studying gene regulatory elements in transgenic mice. Moreover, with similar methods of targeting human ES cell lines (refs) now emerging there is further scope to replace a greater number of transgenic animals with ES cell based differentiation assays. In addition to reducing the numbers of animals used in this type of research the proposed studies will help identify key lineage specific regulatory elements. Such elements in turn hold great potential to drive the development of novel in vitro differentiation protocols which will be vital to harness the promise of embryonic stem cells for regenerative medicine.
This proposal therefore addresses important issues both in fundamental biomedical research as well as developing technologies with the potential of significant replacement of animal experiments.