DE NOVO ES cell isolation
Embryonic Stem cells represent an essential tool for many researchers. Undifferentiated ES cells are necessary for the generation of genetically mutated mice, and for the differentiation into different lineages to test gene function in cell culture systems instead of or in parallel to in vivo studies.
The service was set up by CFCM in collaboration with Giacomo Consalez head of the Developmental neurogenetics Unit, IRCCS Ospedale San Raffaele.
In CFCM protocol, blastocysts are recovered and allowed to attach to a substrate of mouse embryonic fibroblasts for 4 days. Then are trypsinized when necessary using different trypsin concentrations in order to favor the growth of undifferentiated colonies. Isolated ES colonies are then analyzed by immunofluorescence using markers known to be specifically expressed in undifferentiated ES cells.
On demand, CFCM injects isolated clones into blastocyst in order to test the germ line transmission of the mutation.
What CFCM staff needs:
- 6 males, 2-4 months old carrying the mutation of interest;
- mutant females of the right age for superovulation (based on the need).