Embryonic Stem Cell Transfer - Embryonic stem (ES) cells 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 cultured 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 by

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 chimaeric.

The pluripotent ES cells can give rise to germ cells as well. Therefore, pure transgenic mice can be recovered from the chimaeric mice using a suitable breeding scheme. Since DNA integration in genome is random, the frequency of targetted gene transfer, i.e., integration of transgene at a specified site, is discouragingly low.

The ES cell line technology, permits the selection of clones(s) having targetted gene transfers from among several clones showing stable transfection; these can then be used to produce transgenic animals having targetted gene transfer.