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Gene Targeting in Mouse ES Cells


 

Services Provided by the Core

The Bioengineering Core will electroporate gene targeting vectors (provided by the investigator) into embryonic stem cells (ES cells). ES lines derived from different mouse strains are available for this service (e.g. 129SV, C57Bl/6, 129SV/C57Bl/6 Hybrid). We usually isolate 288 antibiotic-resistant ES clones (three 96-well plates), which will be frozen along with duplicate plates for DNA isolation. Due to the nature of the experiment, we cannot guaranty a specific number of antibiotic-resistant ES clones. The identification of recombinant ES clones (e.g. by PCR, Southern blotting) is the responsibility of the investigator. The Core will provide assistance in designing gene targeting strategies and screening strategies for ES clones (contact us for details). Once targeted ES cell clones have been identified and confirmed to have a normal karyotype, we will schedule a blastocyst injection to generate chimeric mice. 

mouseCellColony.jpg

 

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Mouse ES Cell Colony 
Source: International Society 
for Transgenic Technology​


Responsibilities of the Investigator

  • Discuss experimental design of the gene targeting experiment with the Bioengineering Core’s facility director or team members (required for all new projects).
  • Provide a detailed map of the gene targeting construct including size, region of homology, selection strategy, and relevant enzyme cleavage sites.
  • Provide 30ug of purified and linearized gene targeting vector DNA (a detailed protocol describing vector preparation is available upon request).
  • Provide documentation regarding the purity, integrity and concentration of the vector DNA.
  • Please submit a completed ES Cell Service Request Form (provided upon request) to the Bioengineering Core along with your purified DNA. 
  • Please note that frozen ES cells in 96-well master plates will be stored for 2 months while clones are being screened and analyzed. These cells will be amplified and used to generate mice once recombinant ES clones have been identified.