We offer a comprehensive genome engineering resource from design to model production, generating mice that meet researchers’ requirements.
We establish and develop new methods of genome engineering for the production of validated mouse models on controlled genetic backgrounds.
To request our services, please complete the Mouse model generation Resource Enquiry form.
RGEN/CRISPR mediated mutagenesis
Typically, a genomic sequence is targeted in one-cell embryos by a Cas9 or Cas9 D10A/sgRNA complex.
The resulting DNA cut is repaired by the cell machinery through NHEJ, giving rise to a deletion or, if a donor DNA was co-delivered, through homologous recombination. The resulting mice are mated and the targeted mutation is transmitted to offspring.
We have established a range of methods, including the use of oligonucleotides or longer single stranded DNA donors and vary reagent delivery method (electroporation, microinjection in the pronecleus or the cytoplasm) for best efficiency according to the desired alleles.
We deliver extensively validated alleles on a variety of genetic backgrounds according to the needs of service users. We assess animals for additional random integration when a donor template is employed.
Gene targeting – generation of complex alleles
We employ this more classical method for the alleles that are not yet accessible directly in the embryo through genome editing technologies.
ES cells are electroporated with a targeting construct and colonies isolated. The clones are screened, amplified, quality controlled and converted to mice by microinjection into blastocysts. Chimera are obtained and mated for transmission of the genetic modification.
We combine classical methods with current advances, employing transient expression of CRISPR/Cas9 reagents to enhance targeting efficiency for challenging projects.
We generate classical targeting vectors or employ varied targeting constructs from external sources, e.g. EUCOMM/KOMP collection to BACs.
New alleles are extensively validated, combining classical methods (i.e. Southern blot) with new technologies (i.e. ddPCR).
ES cell to mouse conversion – blastocyst microinjection
Mutated ES cells can be obtained from a variety of sources.
The cells are imported, amplified, quality controlled and converted into mice following blastocyst injection. Chimera are obtained and mated for transmission of the genetic modification.
Alleles are extensively validated, combining classical methods (i.e. Southern blot) with new technologies (i.e. ddPCR).
Find mutated alleles by searching the IMPC web portal.
Pronuclear injection – transgene delivery
A DNA construct can be injected into 0.5-day-old fertilised embryos to produce transgenic mice containing the DNA in every cell.
The DNA construct typically consists of a gene of interest that is expressed in a certain range of tissues. The resulting mice are mated and the DNA construct is then transmitted to offspring.
We have experience with employing a broad range of construct types: including plasmids, BAC, PAC.
Please contact us with enquiries on specific genetic background.