Gene targeting is the traditional technique utilizing homologous recombination between an engineered exogenous DNA fragment and the genome of the mouse embryonic stem (ES) cells. These modified ES cells can be injected into mouse blastocysts to generate chimeras carrying the targeted alleles.
Labs have the option of generating targeted ES cells in their own lab (Targeting guidelines) or purchasing targeted cells from repositories.
Depending on your needs, the Genome Editing and Animal Models core is able to perform some of the steps required: planning and construction of gene targeting vectors, targeting ES cells or blastocyst injection.
Gene Targeting of ES cells
There are many standard protocols for targeting. Here is one: https://www.komp.org/protocols.php
1. Verify parental cell lines used for targeting (129/Sv, modified B6 ((JM8A3, JM8A3.N1, JM8A1.N3)
2. Obtain at least two targeted clones with verified correct 3’ and 5’ sequences of regions of homology included in the targeting vector.
3. Clones test mycoplasma-free
4. Clones are karyotyped as normal (Wicell does mouse Karyotyping)
Microinjection of ES Cells
1. GEAM recommends two days of microinjection/clone.
2. Clones may be provided in frozen vials at least two weeks prior to injections for GEAM to expand/microinject or provided on the day of microinjection as single cell suspension.
3. Ten-twenty single cells are injected into each expanded blastocyst. Following the microinjections, blastocysts are transferred into the oviducts of pseudopregnant recipients. Pups are born 17 days later and coat color can be identified one week after that. The founder chimeras will be ready to mate to C57BL/6 partners at 6-7 weeks of age. Up to three chimeras from each clone will be bred in the GEAM. Any additional chimeras can be shipped to the investigator for breeding.
ES cell clones are stored in GEAM. A $95.00 yearly liquid nitrogen storage fee is charged per Principal Investigator.
Investigators can request their ES cell clones at any time and ES cell clones will be processed according to the Principal Investigator wishes.
ANIMAL AND EMBRYO SERVICES
One microinjection day of purchased targeted ES cells into C57BL/6J blastocysts