We assist WIMM researchers in the design and assembly of targeting constructs in the process of generating transgenic animals. We design Southern strategies for screening of targeted ES cells, provide general construct design and subsequently perform custom made assembly of targeting vectors as well as compound and BAC-transgenic constructs. We are using the lambda Red/ET recombineering pipeline that allows targeting DNA molecules at any given position without depending on restriction enzymes. Definition of the homology arms decides over retrieval of DNA into a custom backbone or insertion of cassettes into BAC or plasmid backbones (see also Figure 2).
As well in the era of CRISPR/Cas9 genome engineering, generation of complex KI alleles requires pre-assembled DNA-based cassettes that will either serve as repair templates or as bona fide targeting constructs. Targeting efficiencies in cell lines are promising, however, when Cas9 RNA guided nucleases (RGNs) are co-injected with their cognate sgRNAs into mouse oocytes, the rate of proper HDR is still very inefficient. To be able to generate mouse models in a timely and cost-effective manner, we offer a Cas9-assisted mESC-targeting pipeline, in which Cas9 creates a cut in the mouse genome and circular targeting constructs (TCs) serve as repair templates.
Service: Generation of Targeting Constructs
- Conceptual design and assembly of targeting constructs: knock-out, conditional knock-out, knock-in and conditional knock-in targeting constructs, compound transgenic vectors, BAC transgenic constructs and general custom designed cassettes.
- Development of screening-strategies for targeted cells.
- Actual screening by LR-PCR and Southern hybridization.
- General help in cloning and recombineering technologies.
- Maintenance of bacterial stocks and of a cassette collection for all intermediates and readily assembled constructs available to WIMM researchers.
Service: General Red/ET applications
- Subcloning of genomic fragments from bacterial artificial chromosomes into customer defined plasmids by recombineering (“gap-repair”).
- Subcloning fragments from plasmid to plasmid by recombineering.
- BAC-shaving: removal of unwanted fragments in BAC clones.
- Removal of LoxP/FRT/rox flanked sequences by bacterial Cre/Flp/Dre expression.
- Insertion of functional cassettes into any chosen position in a BAC or plasmid backbone