Gene targeting in ES cells
The Core performs gene targeting experiments in E14Tg2.a and JM8.N4 ES cells. The Investigator provides 100 micrograms of linearized targeting construct and documentation about targeting vector design, preparation of linearized DNA, and selection markers used in the targeting construct. The targeting construct will be transfected into ES cells provided by the Core. Approximately 4 weeks after transfection, the Investigator will receive genomic DNA samples from not less than 250 selected ES cell clones. Screening for targeted ES cell clones will be performed by the Investigator. If compatible with the experimental design, controls for efficacy of the selecting process will be provided by the Core. The Core will retain frozen ES cell stocks until completion of the screening process. The Core commits to providing the above number of selected ES cells, but cannot guarantee successful gene targeting.
The Core performs gene targeting experiments routinely in germline-competent E14Tg2.a and JM8.N4 ES cells with selection in G418 and Gancyclovir, but other cell lines and selection procedures are available upon request.
For Investigators using the E14Tg2a.4 cell line for gene targeting and/or blastocyst injection, you must agree to and sign the "Conditions of Use" agreement from the Mutant Mouse Regional Resource Centers (MMRRC) which can be obtained from the Core Supervisor. This agreement includes acknowledgement of BayGenomics, which donated the E14Tg2a.4 cell line to the MMRRC for distribution, and the MMRRC in any presentations and publications reporting use of the cell line.
For Investigators using the JM8.N4 cell line for gene targeting and/or blastocyst injection, you must agree to acknowledge UC Davis in any resulting publications.
To initiate an experiment, the Investigator contacts the TG Core Supervisor and submits a completed order form for gene targeting. Experiments will be performed at the earliest possible time, usually beginning within 4 weeks after receipt of the DNA construct. DNA for screening will be made available approximately 4 weeks thereafter. Fee information is also available from the Core Supervisor.
Additional services for gene targeting experiments such as expansion of ES cell clones, analysis of modal chromosome numbers, derivation of homozygous recombinant ES cells by selection in high concentrations of G418, and preparation of feeder-cell free ES cells can be provided at the request of, and in collaboration with, the Investigator.