Fig. 4

Production of transgenic animals via the injection of the recombinant DNA into the pronucleus of a fertilized ovum (Method I) or the injection of transformed embryonic stem cells that contain the recombinant DNA into a blastocyst (Method II). In the first and most common method used for livestock species, an ovum is surgically collected shortly after its fertilization, and a recombinant DNA (e.g., the plasmid DNA of interest) is then microinjected through a very fine needle into the pronucleus of the fertilized ovum. The transformed ovum is developed into a blastocyst in vitro and the embryo is then transferred into a surrogate mother for development to term. In the second method, established embryonic stem cells (prepared from a preimplantation embryo) expressing the gene of interest in their genome are transfected with a recombinant DNA. Stably transformed ES cells are selected and then injected into the inner cell mass of a recipient blastocyst. After a short period of culture, the embryo that contains the recombinant gene in its genome is transferred into a surrogate mother for development to term. In both methods, surrogate mothers produce transgenic offspring, but the origin of the blastocyst differs