Generation of Rosa26 Knock-in mice
The analysis of transgenic models generated by pronuclear microinjection is frequently complicated by the undesirable consequences of random integration. Genomic sequences flanking the insertion site can silence or lead to ectopic transgene expression and endogenous sequences can be disrupted confusing the resulting phenotype. Individual founders have a unique site of integration and have a unique number of integrating constructs, necessitating the breeding, characterisation and analysis of multiple independent transgenic models per construct.
As an alternative, transgenic constructs can be precisely inserted into positions within the genome in a controlled and precise manner using ES cell technology. The Rosa26 locus on chromosome 6 is a well characterised neutral locus which is frequently used as a docking site for transgenic constructs. Expression cassettes can be inserted within the locus so that they are expressed ubiquitously from the endogenous Rosa26 promoter or exogenous promoter elements can be introduced to drive the transgene expression in a particular tissue type.
This technology is particularly appropriate when transgenic models are to be compared, for example to establish the functional consquences of sequence mutation or polymorphism. Genetically modified mice can be generated which contain variant transgenes precisely located within the Rosa26 locus - the resulting models can thus be compared with high confidence avoiding the complication of position effects.
To introduce sequences into the Rosa26 locus, we will use a PhiC31 integrase mediated cassette exchange methodology which is much faster and more efficient than conventional gene targeting. Transgenic constructs are cloned into a shuttle vector and exchanged with genomic sequence at the target locus. Recombinant ES cells are then injected into pre-implantation embryos to generate chimeras which can be bred to establish the required genetically modified mouse line.
- Insertion of the transgenic construct into the Rosa26 shuttle vector
- PhiC31 integrase mediated cassette exchange in ES cells
- Screening of recombinant clones
- ES cell injection into pre-implantation embryo injection
- Generation and housing of chimeras for up to 3 weeks.
- Recommendations for breeding strategy.