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Injection
Generating a chimera capable of being bred to heterozygosity or homozygosity is instrumental to establishing a genetically modified mouse model. This is accomplished via the injection of targeted embryonic stem (ES) cells into a donor embryo from an appropriate background mouse strain. If there is sufficient targeted ES cell contribution to the chimera, the gametes will contain the customized genetic information and pass on the targeted allele to subsequent progeny through germline transmission.
Two microinjection motifs for the production of chimeras are currently employed. The first is blastocyst injection, where a 128-cell stage embryo or blastocyst is injected with targeted ES cells. These ES cells then go on to contribute to the differentiating and developing mass of cells known as the intracellular mass (ICM). The subsequently developed chimera has a varying degree of ES cell contribution.
The second type of injection involves the introduction of targeted ES cells into a much earlier, 8-cell stage embryo. The cellular environment of this 8-cell embryo contains more developmental plasticity or potential. Because the ES cells are being introduced into the embryo at a much earlier stage, using microinjection with a laser ablated hole in the zona pellucida, the contribution of the ES cells is more substantial. This type of injection increases the chances of germline transmission. It is also possible that this technique could yield a chimera that is effectively completely ES cell derived.
