EFucci an atlas of proliferation to reduce mouse usage in embryonic proliferation assays

Proliferation describes the process of growth and division of individual cells within a tissue in order to collectively increase its size during development or maintain its size once it is established. It is key to embryonic development and physically drives the growth of the embryo and the formation of tissue subtypes and organs. Regionalised changes in proliferation rates can help organs to develop and drive the determination of their shape or structure. For these reasons the study of proliferation is fundamental to almost all research groups who focus on embryonic development in the context of health and disease and also in many other contexts. There were over 3000 academic papers published in the field in 2013 alone. To mark dividing cells mice can be administered substances that integrate into their DNA during the cell division process, these animals can then be killed and the pattern of cells positive for these markers can be investigated. This experimental approach involves multiple injections and/or the surgical implantation of devices to slowly release these substances. We have developed an alternative mouse model to label dividing cells that does not require any surgical interventions. The purpose of this grant is to use this new model to create an atlas of embryonic development that allows research groups to better design and plan their proliferation assays and so reduce the number of mice they would require to carry out their studies. The atlas will also help to promote the use of our alternative model thus reducing the number of surgical procedures required to carry out the experiments.

The study of proliferation is key to the characterisation of many models of human disease especially in the developing embryo. There were over 3000 academic papers published in the field in 2013 alone. Often these studies use the incorporation of thymidine analogues into the DNA of dividing cells to identify sub-populations of cycling cells. This approach usually requires either multiple intraperitoneal injections of the thymidine analogue or the surgical implantation of mini osmotic pumps to slowly release the analogue. The Fucci system is an alternative to this approach that uses two genetically encoded biosensors of cell cycle phase to label cells and therefore does not require surgical intervention. We have developed an alternative mouse model that incorporates the Fucci probes into a single construct driven by a strong ubiquitously active promoter (R26Fucci2a). This has allowed us to reduce animal usage in our own experiments by abrogating the need to use two mouse lines, one expressing each Fucci probe. #The atlas will also help to promote the use of our alternative R26Fucci2 model thus reducing the number of surgical procedures required to carry out the experiments.

This grant application is for pilot funding to establish an embryonic atlas of proliferation (EFucci). By exploiting a new mouse model of proliferation we have developed (R26Fucci2a), this atlas will serve to directly replace some animal experiments with web based exploration of a large dataset of embryonic proliferation data. The dataset will also allow researchers to obtain pilot data in order to, for example, perform power calculations helping them to design better experiments or identify the critical time points on which to focus their experiments. It will also serve indirectly to promote the R26Fucci2a model we have developed to reduce the dependence on traditional BrdU based cell proliferation assays.

Direct Impacts of the EFucci Atlas:

1). Reduction: In the number of animals required to study the cell cycle by better experimental design
2). Replacement: Of baseline experiments required for experimental design and for wild type developmental studies

Indirect Impacts by promotion of the R26Fucci2a mouse model:

1). Refinement: A 100% reduction in procedures involving BrdU administration in our own laboratories
2). Refinement: A 30% reduction in the number of procedures requiring injections or mini osmotic pumps globally (representing 1311 procedures per annum)

It is easier to estimate the impact of the indirect effects of promoting the use of our R26Fucci2a mouse model. In our own group we have refined 100% of our proliferation assays by replacing traditional BrdU based techniques with confocal imaging of R26Fucci2a animals. If there is a 30% uptake of the method in groups that have previously used thymidine analogues we estimate that 1311 procedures could be avoided per annum.