Gene Targeting

Embryonic stem (ES) cells will be electroporated with targeting vectors provided by investigators. Instructions for the preparation of targeting vectors for transfection can be found here.  Transfected cells will be plated and subjected to drug selection. Clones of cells will be picked and replica-plated in 96-well format. One replica plate of cells will be frozen when confluent; DNA will be prepared from the other plate and given to the investigator for analysis. Each gene targeting experiment will provide the investigator with DNA from 100-500 clones of cells for analysis, depending on the nature of the construct, drug selection strategy and the efficiency with which clones are generated. The DNA purification procedure employed by the core is best suited to analysis by Southern blot, but can also be used for PCR screens.

The core facility has experience using a variety of ES cells for gene targeting experiments. The preferred choice at present is the E14Tg2A.4 feeder cell-independent line from 129/Ola mice prepared by Dr. William Skarnes (The Wellcome Trust Sanger Institute, Cambridge, England).  Other ES cells (e.g., C57BL/6 ES cells) are also available, but the costs associated with using them are higher.

ES cell clones identified as interesting by an investigator will be thawed, expanded and refrozen.  DNA will also be extracted from the thawed clones, and this will be provided to the investigator for confirmatory analysis prior to microinjection.  Up to 12 clones will be thawed and expanded for the indicated cost.  Note that the thawed plates will not be refrozen, so clones can only be recovered from a given plate at one time.

Gene-targeted ES cells can be retransfected with Cre or Flp recombinase expression vectors as required by the investigator.  The cells will be replated and subjected to drug selection if necessary.  Clones of cells will be picked, expanded and frozen down.  96-well plates of DNA will be returned to the investigator for analysis.

1-2 days of microinjection are usually required to complete the injection of up to 100 blastocysts with 2-3 clones of ES cells. The injected embryos will be transferred to pseudo-pregnant recipient mice. Implanted dams will be maintained in the facility until their pups can be weaned and delivered to the relevant investigators. 30-50 injected embryos are usually sufficient to generate chimeric mice that are representative of what can be expected for a given ES cell clone. Details concerning the preparation of ES cell suspensions to be used for microinjection are provided here. The core facility will microinject ES cells from the gene targeting experiments it performs; it will also microinject ES cells provided by investigators.

Preparation of DNA for Gene Targeting

Preparation of ES Cells for Microinjection