Services and Pricing

RMCE (Recombinase-mediated cassette exchange)

Methods that were developed in the laboratory of Dr. Magnuson for efficient RMCE in mESCs have recently been transferred to this Shared Resource. His laboratory, which performs gene targeting and RMCE experiments for the Beta Cell Biology Consortium, has generated several different loxed cassette acceptor (LCA) alleles in mESCs. Once made, these mESCs can be used repetitively to insert various cassettes into mESCs containing the cassette acceptor alleles. This technology is now offered by this Resource to interested investigators. Several investigators are currently using vectors developed by the Magnuson Laboratory to design their own LCAs in mESCs.

In addition, Dr. Magnuson has begun providing several of the LCA-containing mESCs to this Resource for use by other PIs.

1. The completed form will be reviewed by both the Managing and Scientific Directors of the resource to ensure that the strategy is solid, and the vector has been correctly documented. If the experiment is judged to be adequately designed and documented it will be scheduled and started as soon as possible.
2. Upon approval of the project, 50 .g of exchange cassette plasmid provided by the investigator is electroporated into mESCs. The cells are then cultured under appropriate positive selection agents (neomycin, hygromycin-B, or puromycin). Surviving colonies are picked and grown in duplicate. One set of colonies is placed in short term storage at -70° and one plate undergoes negative selection. DNA from the colonies surviving negative selection is used for PCR analysis.
3. After screening results are reviewed, and both the PI and Managing Director agree on which clones are potential positives, clones are thawed, expanded and frozen in liquid nitrogen. Additional DNA is prepared from these clones, and given to the PI who is then responsible for confirming that the gene targeting event is correct by performing additional analysis. Because cells are frozen in 96 well dishes, they cannot be thawed and refrozen. For this reason all undesired clones on that 96-well plate are discarded.
4. Upon confirmation of the clones, at least one mESC clone is chosen for microinjection into blastocysts derived from timed matings of C57Bl/6 mice. Chimeric males resulting from the blastocyst microinjection experiments are provided to the investigator following serology testing by the Division of Animal Care. This usually occurs at about 7 weeks of age. The PI is then responsible for mating these animals and reporting back to the resource the number of pups generated from each chimera and germ line transmission results.

Experimental Flow Chart:

1. PI submits DNA and appropriate service form to TMESCSR for electroporation
2. Service forms are reviewed and approved by the Co-Directors of the facility
3. Electroporation is scheduled, cells are prepared for the experiment
4. Electroporation performed, cells are under selection for 5-7 days before picking of individual colonies
5. Colonies are picked, grown in duplicate, one set is frozen
6. Negative selection of growing colonies
7. Analysis of colonies surviving negative selection
8. Targeted clones are grown and expanded and frozen back in LN2
9. Additonal clone DNA is provided to the investigator for rescreening
10. PI confirms correctly targeted clones and requests ES cell microinjections