Manipulation of mouse embryonic stem cells for knockout mouse production

Abstract

The establishment of mouse embryonic stem (ES) cell lines has allowed for the gene?ration of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene-targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies.

Figure 1

Overview of Electroporation and Selection. The left column shows the approximate number of days at each critical step in the preparation of targeted embryonic stem cells. The middle column describes key procedural events. The last column is an illustration of each critical event. Note that in stages II through V embryonic stem cells should remain as close to pluripotent as possible. Once the cultured ES cells have been plated to the tissue culture plate depicted in step V, they may become differentiated. These cells will used for DNA screening as depicted in step VII. In step VII, the left panel represents an autoradiographic film image of a Southern blot. Southern blot is the definitive test for detection of a targeted event. Many investigators first screen all clone DNA samples by PCR followed by confirmation with Southern blot. A strategy for differentiating between the knockout allele and the wild-type allele should be worked out in advance of initiating a targeting experiment.

Figure 2

Mitotically inactivated ES cell feeders and PMEF. Panel A is primary mouse embryonic fibroblasts that have been treated with Mitomycin C. The treatment arrests the cell division and produces cytokines, which inhibit differentiation of ES cells. Note that the cell bodies appear swollen and resemble a cobblestone-like appearance. Panel B shows PMEF that have not been treated with Mitomycin C. These fibroblasts have a elongated and sickle-shaped appearance.

Figure 3

Panels A and D are images of embryonic stem cells taken with phase contrast filter. Panels B and C are bright light images. Panel A shows embryonic stem cells under drug selection pressure. The far right colony appears to be surviving drug selection (note the sharp, bright border. The other two colonies (rough border) probably would not survive drug selection. Panel D shows a high magnification of a good embryonic stem cell colony. The border appears very sharp and the mass of embryonic stem cells within it appears indistinguishable from each other.