Generation of Knock-out / Knock-in mouse lines
Gene targeting in embryonic stem cells
Through the process of gene targeting in ES cells, potentially any genetic desired genetic manipulation can be introduced into the germline of mice. Specific gene sequences can be deleted either constitutively or conditionally (Knock-out), specific mutations or base pair alterations can be made precisely within a gene of interest and reporter genes or other expression cassettes can be introduced into defined locations within the genome (Knock-in). Murine sequences can be exchanged for human sequences using this technology to generate humanised mouse models.
This technique can take up to a year of development time so it is essential to invest considerable time at the beginning of the project to ensure that the most appropriate technology and genetic strategy are selected for the required model. The transgenic core can provide consultancy for these types of decision and it is essential that all targeting projects be discussed in detail with the core before commencing work in the laboratory.
When a strategy has been decided upon, a targeting vector is constructed and a robust screening strategy is devised and tested to ensure that the required genomic manipulation can be detected at the genomic level. The targeting vector is then transfected into ES cells and recombinant clones are screened for the desired homologous recombination event. These cells are then injected into pre-implantation embryos and the resulting chimeric mice are used for breeding the genetically modified mouse line.
A flexible service is offered which can be broken down into the specific stages of model development, depending on the available resources.
Project Initiation - Free of Charge
- Consultancy with the group to address an appropriate technology to achieve a safe, usable model
- Screening of gene trap databanks for pre-existing ES cell clones
- Screening of Knock-out consortium resources for pre-existing targeting vectors, ES cell clones and models
Consultancy for targeting vector construction - Free of Charge
- Genomic sequence analysis to identify regions which have a high probability of being usable homology regions for gene targeting
- Consultancy with the group to address the genetic strategy required to achieve a safe, usable model
- Construction of the targeting vector is not provided by the core but the core can assist with the provision of cassettes and methodology advice
- Establishment of the screening strategy is not provided by the core but the core can assist with the provision of cassettes and methodology advice
Gene Targeting in Embryonic Stem cells
- Transfection of the completed targeting vector into embryonic stem cells
- Isolation and storage of at least 192 independent recombinant cell clones
- DNA preparation and delivery of 96 well plates for screening
- Thawing, expansion of putative positive clones
- Additional DNA preparation from putative positive clones for confirmation screening
- Karyotype analysis of the targeted clones
Blastocyst Injection of ES cell clones
- ES cell injection into pre-implantation embryo injection
- Generation and housing of chimeras for up to 3 weeks.
- Recommendations for breeding strategy