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Home » Biology Homework Help » Biotechnology » Embryonic Stem Cell Transfer
Embryonic Stem Cell Transfer
Embryonic stem (ES) cells of mice are pluripotent cultured cells derived from early pre-implantation embryos. These cells can be maintained and multiplied in vitro long enough to permit the various manipulations for gene transfer. The ES cell technology cells technology may be described in simple terms as follows. The cultured pluripotent ES cells are transfected with the appropriate transgene construct by a suitable transfection technique. Transfected ES cells are identified and selected, generally by employing a selectable marker gene, and are cloned.

The production of transgenic animals using transfected ES cell clones may be achieved in one of the following two ways: (1) injection, and (2) coculture. The cloned ES cells are injected into the blastocoels of blastocyst stage embryos; the blastocytes are obtained from donor females in a similar manner as that for microinjection. Alternatively, the zona-pellucida of 8-cell to morula stage embryos is removed, and the morulae are co-cultured with the ES cells; the ES cells are preferentially incorporated into the inner cell mass of the developing embryo. It is also possible to transplant the ES cell nucleus into an enucleated fertilized ovum (an ovum whose nuclei have been removed), but this technique is not commonly employed as it is quite tedious although all the progeny obtained by this technique are transgenic.

The embryos cocultured or injected with transfected ES cells are transferred into surrogate mothers where they complete their development. About 30% of the progeny derived from such embryos contain tissues derived from the ES cells, i.e. they are chimeric. The pluripotent ES cells can give rise to germ cells as well. Therefore, pure transgenic mice can be recovered from the chimeric mice using a suitable breeding scheme.

Since DNA integration in genome is random, the frequency of targeted gene transfer, i.e. integration of transgene at a specified site, is discouragingly low. Therefore, transfection techniques like microinjection are not suited to the targeted gene transfer. ES cell line technology, on the other hand, permits the selection of clones(s) having targeted gene transfers from among several clones showing stable transfection. The desired clone can then be used to produce transgenic animals having targeted gene transfer.

ES cells technology is at present confined to mice because embryos of most other mammals do not survive in vitro so that it is extremely difficult to produce and maintain their ES cell lines. Even in case of mice, ES cell lines rarely give rise to germ cells in individuals of some strains making it virtually impossible to produce transgenic animals of these strains using this technology. Recently, ES cell lines have been developed in some mammalian species other than mice, but so far chimeric individuals have not been produced using them. It may be hoped that in the near future it may become possible to apply this technology in some mammalian species.

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