Gene Targeting in Embryonic Stem Cells

Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

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.

Abbreviations

2-ME

Beta-Mercaptoethanol

DMEM

Dulbecco’s Modified Eagle Medium

DMSO

Dimethyl sulfoxide

D-PBS

Dulbecco’s phosphate buffered saline

ES cells

Embryonic stem cells

FBS

Fetal bovine serum

FIAU

1-(-2-Deoxy-2-fluoro-1-b-d-arabino-furanosyl)-5-iodouracil

LIF

Leukemia Inhibitory Factor (ESGRO ®)

MEF

Mouse embryonic fibroblast feeder cells

MEM

Minimal Essential Medium

Pen/strep

Penicillin/streptomycin antibiotic mix for cell culture

SDS

Sodium dodecyl sulfate

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Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  1. 1.University of Michigan Transgenic Animal Model CoreAnn ArborUSA
  2. 2.Transgenic Animal Model CoreUniversity of Michigan Medical SchoolAnn ArborUSA
  3. 3.Division of Molecular Medicine and Genetics, Department of Internal MedicineUniversity of Michigan Medical SchoolAnn ArborUSA

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