Transgenic Animals

To get the targeted gene into the mouse genome, Capecchi used a very specialized embryonic cell previously isolated by Matthew Kaufman and Martin Evans. These embryonic stem (ES) cells were isolated from an early stage of embryonic development (the blastocyst). When grown under controlled conditions in culture dishes, the ES cells have the remarkable capacity to become cells belonging to any tissue type. They can become muscle, cartilage, blood vessel or nerve cells, for example. Even more astonishing, when ES cells are injected back into a blastocyst, they mix with the cells of the recipient embryo and contribute some cells to every tissue in the body. Thus, if researchers place a transgene into the ES cell's genome and inject those ES cells into a blastocyst, the transgene could end up in every tissue type of the mouse.

Typically, the targeting vector is placed into ES cells derived from a mouse that has a brown coat. ES cells with the targeted gene replacement are identified through positive and negative selection. They are then injected into blastocysts from mating mice with black coats. These blastocysts are then placed into a surrogate mother and allowed to develop to term. The offspring that have incorporated transgene-containing ES cells into some of their tissues are identified by having patches of brown coat color.

To allow the targeted gene replacement to be passed to subsequent generations, the ES cells must also contribute to the developing embryo's eggs or sperm. To determine if the targeted gene has been incorporated into a mouse's eggs or sperm, the mice with brown patches are mated to black-coated individuals. The brown coat color is a dominant trait, so any off-spring with brown coats can be assumed to have arisen from germ cells that derived from the manipulated ES cells and thus contain the targeted gene replacement. These mice are mated to brown-coated siblings to produce homozygous transgenics, which are identified by determining if the offspring contain two copies of the transgene replacing both normal copies of the gene in the genome. These homozygous mutants are studied to look for phenotypic changes due to the transgene.