Knock-out/Knock-in Mouse production
Targeted mES cells are injected into blastocyst stage embryos which are then transferred in pseudopregnant females to develop to term. Mouse strain routinely used as blastocyst donor is C57Bl/6N. However, other mouse strains are also available. From the resulting chimeric mice the degree of chimerism is evaluated based on coat color or PCR. The best chimeric males are bred with C57BL/6 mice in order to transmit the mutated allele through the mouse germline. Blastocyst injection service includes injection and transfer of approximately 60 blastocysts. We also provide service in following steps of chimera breeding.
TCDM has the expertise for tetraploid ES cell aggregation that is used to generate embryos that are completely ES cell derived. With the technique one can e.g. rescue an embryonic lethal phenotype caused by a placenta defect.
CRISPR-Cas9 editing in zygotes
TCDM will perform the microinjection or electroporation of gRNA/template DNA/Cas9 protein mix and following embryo transfer. Mouse strains available are C57BL/6N and FVB/N. We also provide service in founder genotyping and breeding.
Transgenic mice are generated by injecting cloned DNA into zygotes which are then transferred in pseudopregnant females to develop to term. Mouse strains routinely used as zygote donors are FVB/N and C57Bl/6N. Genotyping samples are taken for screening potential founders by PCR or southern blot. A robust screening method must be established before submitting DNA for microinjection. Service includes purification of the DNA construct, microinjection, transfer of 300 embryos and taking genotyping samples.