Advanced Protocols for Animal Transgenesis

Part of the series Springer Protocols Handbooks pp 291-325


Gene Targeting in Embryonic Stem Cells

  • Elizabeth D. HughesAffiliated withUniversity of Michigan Transgenic Animal Model Core
  • , Thomas L. SaundersAffiliated withTransgenic Animal Model Core, University of Michigan Medical SchoolDivision of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School Email author 

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Genetic modification of mouse embryonic stem cells is a powerful method to study gene function in whole animal models. The ability to re-design genes in mouse to reproduce genetic defects found in human patients gives researchers a wide open arena for biomedical research. Successful manipulation of ES cells requires culture conditions that restrain differentiation and support robust growth. It is essential to use culture conditions that are carefully calibrated to maintain ES cells in pluripotent condition because the only test for germline transmission is the time-consuming process of preparing ES cell-mouse chimeras and breeding them for several months. Fastidious adherence to culture conditions will maintain the capability of ES cells to differentiate into many cell types. Inadequate care of ES cells will degrade the ES cell quality and result in cells that fail to transmit mutant genes through the germline. Mouse ES cell culture is a well-established procedure. After targeting vector electroporation and identification of euploid gene-targeted clones, generation of chimeras from three clones is normally sufficient to transmit engineered mutations through the germline and produce a new mouse strain. Although the process of generating a new mouse model is not always routine, careful attention to ES cell culture is rewarded by the production of robust gene-targeted ES cell clones that regularly go germline.