Embryonic Stem Cell Injections into Mouse Blastocysts
General information
Gene targeting is the process of disrupting or mutating a specific genetic locus in
embryonic stem (ES) cells, usually with the intention of making knock-out or knock-in
mice. These ES cells are then injected into host blastocysts collected from mice with a
different coat color than the ES cell donor strain. The ES cells integrate into the embryo
and can contribute to all tissues of the developing chimeric mouse. The injected
blastocysts are surgically transferred to pseudopregnant foster mothers.
We offer gene targeting in 129 (CJ7), C57Bl/6 (AgoutiJM8.N3) and a hybrid of both line
129 and C57Bl/6 (EC7.1) ES cells. All of our ES cell lines have demonstrated good
targeting frequency by homologous recombination and proven germline transmission when
injected into mouse blastocysts.
There are various advantages and disadvantages of targeting the above ES cell lines.
Traditionally 129 ES cell lines are used because they can be cultured easily and they give
rise to high percentage chimera. The disadvantage is that the chimeric mice are a genetic
mosaic of 129 and C57Bl/6 cells and require many generations of back crossing to
generate a pure Bl6 background. On the other hand, targeting in the C57Bl/6 ES cell line
is just as efficient, and the chimeric mice are in the same genetic background and would
not require back crossing. We encourage you to consult with our TGTC staff in choosing
the right approach for your experiment. Please email us at makeamouse@ucdenver.edu.
We can visualize ES cell contribution to the development of the chimeric mouse by coat
color mismatching. Following birth of the chimeras, they are scored to determine the
percentage of ES cell contribution (or percent chimera) by estimating the amount of coat
color contributed by the ES Cell genome and the coat color contributed by the blastocyst
donor strain. A 100% chimeric mouse would be the color of the mouse strain from which
the ES cells were derived.
Info es cell lines v2.5.doc sw/ja/pjk 7/16/2010