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Microinjection of embryos
DNA, RNA, Proteins, RNP’s are microinjected into 50-100 fertilized single-cell (or two-cell embryos), which is generally enough to yield 10-20 live births. Injected embryos are then surgically transferred into the oviduct of pseudo-pregnant females. Pups are typically born 19 days after transfer. Pregnant females are followed closely and foster mothers are available if problems occur. Occasionally, litters are lost due to cannibalism. If litters are lost, additional injections can be scheduled immediately.
The technology of CRISPR/Cas9 is a new and fast method for generating mice with a mutation, deletion or insertion in a gene at a defined position within the genome. For CRISPR/Cas9, the desired Cas9 protein, sgRNAs and if required DNA or ssDNA template will be injected into the pronucleus of zygotes or two-cell embryos. For the generation of KO mutations, we are using an electroporation method. We have successfully used this technology to create the mutations directly in single-cell fertilized embryos:
- KO mutations
- Single point mutation KI
- Insertion of epitope tags to create tagged fusion protein
- New mutations in established mutant mouse lines
Investigator-provided ES cells are injected into the cavity of an expanded blastocyst stage embryo (CD1 or C57Bl6) and injected embryos are surgically transferred into the uterus of recipient females.
Not all ES cell clones will result in chimeric mice and not all chimeric mice will transmit the mutated allele through the germline to the next generation. The ability to generate germline transmitting chimeras is dependent on the pluripotent state of ES cells. For these reasons, we cannot guarantee that microinjections of stem cells will result in the generation of chimeric mice or that chimeric animals will be germline competent.
We have successfully generated germline competent chimeras from investigator-supplied targeted clones. We offer to inject the same ES cell clone in blastocyst stage embryos on two consecutive days to ensure enough surviving pups.
We also offer aggregation of ES cells with morula stage embryos and tetraploid complementation assays.
For full-service packages and ES stem cell work we are collaborating with the MutaMouse genome engineering unit.
Cryopreservation of mouse lines
It is a safe method for preserving healthy pre-implantation embryos or sperm that can be easily recovered to revitalize the strain at any time.
There are many reasons to consider cryopreservation of your mouse lines:
- Insurance against loss of colonies in the event of calamity.
- They are a back-up in case of reproductive failure within a colony.
- Results in significant long-term cost savings and mouse room space.
- It helps to control genetic drift
- A genetic resource for refreshing mouse lines.
- Cryopreserved stocks can be shipped to or from collaborators worldwide
Sperm cryopreservation is a simple, highly cost-effective method for cryopreservation. Although only half of the genome is preserved as frozen sperm, cryopreservation of sperm is significantly more cost-effective and faster as compared to embryo cryopreservation. This technique works well with common backgrounds like C57Bl6, CD1. We are storing more than 250 strains since 2011. The recovery of sperm from cryopreservation requires an In Vitro Fertilization (IVF) procedure. Our recovery rate after IVF is 94%.
Embryo cryopreservation is the method for preserving mouse lines with a complex genetic background. However, due to inherent technical inefficiencies associated with the procedure and strain differences, it is more costly than sperm freezing. Some strains have low fertilization capacity and do not yield sufficient numbers of fertilized embryos. This could partially overcome by the use of IVF. Our recovery rate is 90%.
We are storing the samples divided over two LN2 containers located at two different buildings on campus.
Revitalization and Rederivation of mouse strains
An important variable that affects the results of mouse studies is the health status of the animals. Pathogen burdens may confound observations and obscure underlying mechanisms. Embryo transfer is the golden standard to obtain specific pathogen-free mice.
Fresh embryos, embryos obtained by In Vitro Fertilization techniques or thawed embryos are subjected to extensive washing procedures and then surgically re-implanted into pathogen-free pseudo pregnant females. The resulting pups will be housed in the Q-zone and will be tested for the absence of pathogens at the age of 8 weeks before sending them to your colony.
The recovery of sperm from cryopreservation requires an IVF procedure. This procedure is typically performed using wildtype oocytes. If desired we can use female animals from an investigator's personal colony. Mouse strains can vary in their sensitivity to superovulation, which can result in low oocyte numbers.
The Mouse Expertise Unit collaborates with The Laboratory Animal Center for Caesarian rederivation. Depending on the initial health status of your strain, Caesarian rederivation is more cost-effective and faster as compared to embryo rederivation.