The Mary Lyon Centre at MRC Harwell is the UK’s national facility for mouse genetics. They work to improve the understanding of pathologies by generating new mouse mutants as models of human disease and for preclinical development. The Centre works with approximately 50,000 mice which allows for the large scale generation of mouse mutants. This presentation outlines classical and modern methods for generating mutants and highlights the integration of digital PCR for validation of these processes.
Gene targeting involves using a targeting construct to electroporate embryonic stem cells (ESLs) and screen for homologous recombination events, resulting in chimeric animals with specific genetic modifications. In contrast, gene editing, particularly using CRISPR/Cas9, allows for direct modification of the genome in one-cell embryos, creating mosaic founders with desired mutations and additional genetic changes.
Teboul guided the audience through the pipeline of genome editing, providing two examples where digital PCR is used. First, how digital PCR can be used to map deletion alleles. Second, the tool is used to validate the generation of desired point mutations or knock in alleles.
After covering genome editing, Teboul highlighted how gene targeting, despite it being a more classical technology, is making a resurgence. This is due to the fact that it can generate more complex alleles than the newer techniques. Teboul discussed how digital PCR can be used to validate this genome targeting, with particular respect to karyotyping and screening targeted materials.
Teboul ended the presentation with a discussion of how digital PCR can complement other approaches. Digital PCR is shown to improve the validation process by allowing for smaller sample sizes and earlier screening stages. Teboul concludes that effective validation requires a multifaceted approach, including sequencing and structural rearrangement assessments.
