ES Cell Electroporation
Gene-targeted mouse ES cells are produced by electroporating a gene targeting construct into wild-type ES cells. The targeting construct typically contains selectable markers surrounded by two regions of homology to the targeted locus. In a fraction of those cells that take up the targeting construct, homologous recombination between the genome of the ES cell and the two regions of homology will result in the replacement of the targeted locus with the targeting construct.
Knock-out or knock-in mice are created by microinjecting these gene-targeted mouse ES cells into mouse blastocysts. The injected blastocysts are then transferred to pseudopregnant females and allowed to develop to term. The resulting pups are chimeras.
The gene targeting service includes the following:
- electroporation of your targeting vector into wild-type mouse ES cells
- culture of cells under antibiotic selection
- picking up-to 96 drug-resistant clones
- splitting clones into two sets of 24-well plates-one set will be frozen at -80°C and the other will be provided to the investigator for genotyping
- thaw and expand targeted ES cell clones
- provide investigator with a 10 cm confluent analysis plate for Southern analysis (one plate for each clone carrying a homologous targeting event)
- freeze and store duplicate clones in LN2 to use for future experiments/microinjections
- In order to ensure that no protein is made from the targeted gene, construct a targeting vector that will delete as much of the coding sequence as possible.
- The arms of homology should be cloned from the same strain as the targeted ES cells. For most projects, we recommend targeting to C57BL/6.
- The longer the arms of homology, the better. A common strategy to aid in genotyping is to design one arm at 5-6 kb and the other arm at 1-2 kb for an overall length of 7 kb.
- It is the investigator’s responsibility to design and engineer the targeting vector. The TRP offers free consultations for targeting vector and genotyping design.
- The investigator should provide 30 µg of linearized targeting construct DNA at a concentration of 1 µg/µl.
- The DNA should be absolutely free of contamination from ethidium bromide, ethanol, phenol, chloroform, and high salt.
- Design a genotyping protocol (Southern Blot Analysis and/or PCR) that can distinguish homologous recombination events from random integration events. This step should be completed before submitting your DNA vector for electroporation. As an aid for establishing a reliable genotyping procol, the TRP highly recommends that all investigators prepare a control vector which includes the area outside of the region of homology.
- G4 ES cells: This line was created by Andras Nagy from a hybrid of 129S6/SvEvTac x C57BL/6Ncr. This is our preferred line for gene targeting experiments and is available to all University of Washington investigators with a signed MTA
- R1 ES cells (129X1/SvJ x 129S1)
- It is the investigator’s responsibility to establish a reliable genotyping protocol (PCR and/or Southern Blot) before the electroporation process is started. Clones must be screened promptly. ES cell clones that are stored at -80° C for more than 1 month will not be guaranteed to produce high-percentage chimeras.
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To schedule an ES cell electroporation session, go to our TRP Forms page. Fill out a “Request for ES Cell Electroporation” form and deliver it along with your gene targeting construct to the Health Sciences Building, room T-140D, during normal business hours. Ask for Serina Tsang.