In vivo inactivation of a gene is possible by the gene targeting technique. The appropriate gene targeting vector is inserted into the embryonic stem cells (ES) and after homologous recombination events the endogenous target gene is inactivated or mutated. Injection of ES recombinant cells into blastocysts give rise to chimeric mice that should be properly crossed to test germ-line transmission of the mutation.
Embryonic stem cell electroporation
The user is instructed and helped in the correct planning of the gene targeting vector by CFCM staff. If the customer already has a vector, CFCM staff require previously to ceck compatibility of your vector to our conditions.
CFCM does not guarantee the presence of recombinant clones.
Some DNA sites appear impervious to homologous recombination, whereas sites a few kilobases away may be much more open to integration. But even at the same site, the frequency of homologous versus non-homologous recombination events can vary by a factor of ten, from one day to the next.
What CFCM staff needs:
- 400 micrograms of Qiagen quality gene targeting vector DNA
- A map of the construct and a map of the targeting strategy
- The restriction enzyme for linearization should be clearly indicated
- A picture of the linearized vector
Embryonic stem cell screening
The screening will be performed by Southern blot. This is the most reproducible technique to screen for recombinant clones.
What CFCM staff needs:
- The customer should provide 400 ng of probe to screen 5’ arm integration and 400 ng of probe to detect the correct 3’ integration
- A picture of the agarose gel of two probes
- A map of the construct and a map of the targeting strategy with the indication of REs to use to cut DNA for the screening
- Indications of the Southern conditions used to test probes (such as hybridization temperature and washing conditions)
Blastocyst injection of targeted ES cells
The C57BL/6 mouse strain used for blastocyst injection is provided and inbred by Charles River or ENVIGO. Usually the ES cell lines derive from agouti or chinchilla/agouti strains and the chimeric pups will be brown or brown/beige on a black background.
CFCM microinjects 10-15 ES cells into 50 blastocysts of C57BL/6, alternatively the staff injects 4-5 ES cells into 50 compacted morulae of the same strain, transfer the embryos into foster mothers and monitor the pregnancies. The pups are maintained at CFCM animal house until weaning and then they are shipped to the investigator.
What CFCM staff needs: if recombinant clones are generated by the customer, he should provide two vials of ES cells ready to be injected and the following data:
- results of mycoplasma test
- cellular morphology description
- cariotype analyses of the clones (this is in the customers interest; can be waived at the customers request)
After injection any remaining ES cells will be destroyed.