- Do you perform pronuclear injection?
Unfortunately, we cannot offer this service for the production of a transgenic line carrying random integration yet, but we can perform pronuclear microinjection of CRISPRs. Also, we can electroporate ES cells with your transgene and generate chimaeras from positive ES cells. The generation of transgenic mouse lines via ES cell electroporation offers some advantages when compared with classical pronuclear injection. For example, normally you can select ES cell clones with only one insertion or check for expression of the transgene in cells prior to blastocyst injection. Please contact us if you want us to advise you on the best strategy for your particular experiment.
- How long will it take to perform an experiment?
This varies, but in general, it will take around 6 months from the ES cell targeting to the production of adult chimaeras. The targeting experiment takes 4 weeks and the generation of adult chimaeras around 3 months.
Rederivation and cryopreservation take around 6-8 weeks to complete. Again, this might vary depending on availability and performance of the mouse line.
ES derivation experiments last around 4-5 weeks from harvesting of the blastocysts to frozen vials of cell clones.
Morula aggregation will only take around 4 weeks from harvesting of the embryos to chimaera production
- How many clones will you pick for each construct?
We will pick around 200-400 clones fro standard targeting via homologous recombination and around 100 clones for CRISPR. After picking we will expand the clones and freeze them. We will also provide you with DNA for the screening experiment. We cannot guarantee that you will get a successfully targeted ES clone. Sometimes a construct is not suitable for targeting and this cannot be predicted in advance.
- What if we do not generate chimeras?
If no chimeric mice are generated after injecting 100-120 blastocysts, we will contemplate the possibility of injecting more targeted clones, if available (i.e., new GeneTrap clones or other clones from our targeting experiment). If the customer has previously paid for the targeting of our ES cells and no chimeric mice have been generated in the first round of injection, we will perform another round of injection free of charge. However, the customer will need to pay for a new targeting experiment in ES cells to generate more targeted ES cell clones.
- Which is the success rate?
So far, we have performed more than 70 targeting experiments in ES cells, of which 56 were successful. The number of ES cell injection projects has been 46 (27 using our ES cells and 19 using ES cells that have been targeted elsewhere). Germline transmission were 23/26 and 14/19, respectively.
- What do I get for my money?
We will do one targeting experiment and one round of blastocyst injections (usually between 100-120 blastocysts). You can apply just for the ES cell work, for the chimera production service or for both. Charges will vary depending on the service(s). Please contact us for more details.