Biomedical Engineering Reference
In-Depth Information
ES cells have optimal growth when slightly crowded. Plate or passage ES cells at a
higher density if it is necessary to increase colony numbers.
4
Lack of chimeras
•
•
After identification of targeted ES cell clones, it is important to minimize time in
culture. We recommend passaging ES cells no more than one to two times from
thawing of frozen stock until blastocyst injection.
•
Chimeras are more easily generated from ES cells with low passage number. It is
preferable to begin ES cell manipulation with as low a passage number as possible.
•
ES cells should be microinjected as soon as possible after collection from culture
dishes, not left on ice for an extended period of time.
•
If troubleshooting attempts have failed due to undetectable differentiation or defects
of the first clone, it may be necessary to prepare a second targeted ES cell clone for
blastocyst injection.
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