Cas9-assisted mouse embryonic stem cell targeting
In a partnership with the Transgenic Core at the Wellcome Trust Centre for Human Genetics we combine our expertise in generating donor constructs for Cas9-assisted homologous recombination with their mouse ESC targeting pipeline for the generation of genetically modified mouse models. We refer to this service as development of a “full mouse package”.
Gene targeting in ES cells mediated by homologous recombination was awarded the Nobel Prize in 2007. Any targeted DNA sequence can be deleted constitutively or conditionally (KO and cKO), and specific mutations or expression cassettes can be inserted precisely within a region of interest (KI and cKI approaches). Until the advent of the CRISPR/Cas9 technology, targeting had to rely on linearized DNA donors bearing negative and positive selection markers. Screening of correctly targeted clones had to discriminate random versus proper insertion by Southern hybridization, with success rates often lower than 1-5%. Using Cas9 to generate specific DSBs directly at the insertion site, we can use circular donors for the homologous recombination process, and thus greatly reduce random insertion events. In concert, the higher rate of proper HR allows to use shorter homology arms, making the first screening amenable for a PCR-based approach. Once confirmed, cells are expanded and injected into pre-implantation embryos. The resulting chimeras are used for establishing the genetically modified mouse line.
Services we offer (in collaboration with the WTCHG)
- Electroporation of circular targeting vector and a Cas9/sgRNA co-expressing plasmid (preferably using a Puromycin selection-marker) into albino C57B/6 mouse ESCs.
- Isolation and storage of routinely 96 independent recombinant cell clones.
- DNA preparation in 96 well formats for initial PCR screening.
- Thawing and expansion of positive clones.
- Additional DNA preparation and confirmation screening by Southern hybridisation.
- Sequencing of inserted components as present in the chosen mESC clone.
- Karyotype analysis of targeted clones (in collaboration with the group of Kath Green, at the Wellcome Trust Centre for Human Genetics).
Services we offer (in collaboration with the WIMM TG Services)
- ES cell injection into pre-implantation embryo injection.
- Generation and housing of chimeras for up to 3 weeks.
- Advice on setting up functional screening strategies to identify targeted animals.
- Recommendations for subsequent breeding strategies.