Cell Biology Services

The Cell Biology Services provides centralized access to all of the facilities, instrumentation, and technical expertise necessary to carry out the complex cell biology protocols required to create genetically engineered mouse models. An assortment of services are offered, including cell line importation, ES cell and primary cell derivation, cell line expansion, gene-targeting (electroporation, drug selection and clone picking), transfection/nucleofection optimization of non-mouse lines, and CRISPR modification of cell lines. Other service offerings include custom projects, training, and provision of tested reagents and supplies. The Cell Biology Service has in its possession several high value and high demand mouse ES cell lines and has been successful in generating genetically modified mice on a variety of inbred strains. In addition, we have extensive experience importing ES cell lines from the KOMP consortium members and generating germline mice using these ES cell lines.

Parental Cell Line:

• C57BL/6J
• 129 S1/SvImj
• C57BL/6N JM8A3
• C57BL/6N PRX B6N #1
• DBA/2J AC173

Electroporation- Approximately 25ug of a DNA construct is transfected into 15 million ES cells using the BTX ECM830 Electroporation system. After electroporation, the ES cells are exposed to drug resistance selection, and the surviving ES colonies are picked into 96-well plates for continued growth. These isolated ES clones are triplicated in 96-well plates. One set is stored at -80 C, and the others are prepared for DNA analysis. After homologous recombinants have been identified, clones from the corresponding 96-well plates are thawed, expanded for stock, submitted for chromosome counts, and DNA isolated for further confirmation.

ES Cell Preparation for Microinjection - Coordination and scheduling of injection of targeted ES cell clones, including expansion and preparation on day of microinjection to thin the MEF cells and re-suspend in hepes buffered media for delivery to the Microinjection Service.

Cell Line Importation Service - All cell lines destined for in vivouse must be tested for the presence of pathogens by the Laboratory Animal Health Services Diagnostic Laboratory. Upon request, the Cell Biology Service will receive your newly-acquired cell line and expand it in our BSL2 laboratory. After expansion, cells will be frozen and a sample sent to the Diagnostic Laboratory for pathogen testing. If cells are pathogen-free, the frozen vials will be released to you or moved to the main Cell Biology Service lab in the GRB for further manipulation.

ES Cell-line, MEF, and Primary Cell Derivations - ES and MEF cells line derivations available for all strains available through JAX Mice and Services or private stains within the investigators laboratory. Bank stock expansion sizes, quality testing, and chromosome counting can all be customized for the researchers needs. Additionally, primary cell derivations are available.

CRISPR Modification of Cell Lines - Modifications of both mouse and non-mouse lines though CRISPR are available, including generation of CRISPR materials and screening with our Molecular Biology Service.

Transfection/Nucleofection of Cell Lines - Optimization of transfection though lipotransfection reagents or Nucleofection is available for both cell lines and primary lines.

FBS Screening

At regular intervals, FBS from several suppliers is screened in order to select inventory that is supportive of ES cell growth. Serum is purchased in bulk to reduce cost for all interested parties.