Title: Efficient ‘On Demand’ generation of complex experimental transgenic mouse models
Funder: Cancer Research UK

Abstract: Background Transgenic mice are indispensable tools that bring relevance to basic and applied research (market size $1.5bn, 7.5% CAGR). Experimental models are traditionally created through targeting alleles in the germline and establishing large breeding colonies to produce experimental animals, prior to commencing experimental work. These approaches are expensive (£20K-£60K for germline targeting) and time consuming (6 mths - 3 years), particularly in cancer biology where multiple disease alleles are often combined. This is incompatible with the pace and scale of modern scientific research. We propose here to bypass germline targeting and breeding, allowing scientists to create experimental animals ‘on demand’. This presents a novel, more ethically palatable approach to in vivo research, that dramatically reduces the time, costs and animal use while allowing faster design-build-test cycles. We aim to identify (Aims 1-3) and exploit (Aim 4) key cell-intrinsic factors that allow a mouse Embryonic Stem cell, maintained in vitro, to create a chimeric animal when implanted to a host embryo. This will allow us to create mice on demand, which are comprised of 90% of mESC-derived cells, ready for use in experiments in as little as 4 weeks. Aims (i) Development of CASpicr, a novel barcoding method that allows isolation of individual barcoded cells from a mixed population (ii) Characterisation of mESC-intrinsic chimeric potential (iii) Development of a bioassay for chimeric potential Methods (i) Development of Cas9-enabled prospective isolation of cells (CASpicr) We will construct a lentiviral-based barcoding library, with barcodes based on CRISPR/Cas9 guide recognition sites. We will use a Cas9 variant as a chemical switch to isolate individual barcoded cells from a pool. (ii) Characterisation of mESC-intrinsic chimeric potential Using a robust in vitro embryonic development assay, we will assess each barcoded mESC for the ability to form a chimera. We will then subject cells with high and low chimeric potential to single cell analysis to identify features that govern chimeric potential (iii) Development of a bioassay for chimeric potential By prioritising feature hits, we will develop a bioassay that will allow the prospective isolation of mESCs that have high chimeric potential. How the results will be used This will allow scientists to reimagine their approach to basic and applied in vivo research, creating only those mice that are needed in an ‘on demand’ fashion, dispensing with the need to maintain breeding stocks, and bringing animal work into the reach of less well funded research establishments.

Lay abstract: Transgenic mice are indispensable tools that bring relevance to basic and applied research. Experimental models are traditionally created through establishing large breeding colonies to produce experimental animals, prior to commencing experimental work. These approaches are expensive (£20K-£60K for germline targeting) and time consuming (6 mths - 3 years), particularly in cancer biology where multiple genetic changes are often combined into one model. This is incompatible with the pace and scale of modern scientific research. We propose here to exploit cutting edge genome editing (CRISPR) technologies to allow scientists to bypass the need to establish large breeding colonies, making only those experimental animals needed for their research. This presents a novel, more ethically palatable approach to in vivo research, that will dramatically reduce time, costs and animal use.

Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY)
Health categories: CANCER AND NEOPLASMS