Cloned genes can be introduced into knockout mice by means of embryonal stem cells (ES cells). Embryonal cells have attracted tremendous attention in recent years, hence a brief introduction on ES cells is warranted. The concept of ES cells arose through studies on mouse and human teratocarcinomas.
These tumours were found to contain cells, called embryonal carcinoma cells, the stem cells of the tumour that were able to differentiate into a variety of adult cells and tissues. Stem cells can proliferate without changing their phenotype and simultaneously change (differentiate) into one or more new cell types.
ES cells were then derived from cells in the inner cell mass of the mouse blastocyst. ES cells can be established in culture. They can also be reintroduced into early embryos where they take part in development and can give rise to cells in all tissues of the mouse, including cells of the germ line. Thus cloned DNA can be introduced into ES cells in culture, the transformed cells can be selected.
These cells can be introduced back into mouse embryos. Such embryos produce chimeric offspring in which some cells are derived from the normal embryo cells, and some from the transfected ES cells. If some such knockout mice have transfected cells in their germ line, breeding these mice allows direct inheritance of the transfected gene in the progeny.
Knockout Mice:
The ability of embryonic stem cells to change (differentiate) into any cell type of the adult organism clearly demonstrates that they are totipotent. In various kinds of studies we come across phenotypes in mice that lack a functional copy of a particular gene.
These animals are called knockout mice and serve as a research tool for gaining insights into mechanisms underlying various cellular activities in which the product of a particular gene is directly involved, as well as in genetic basis of a human disease. Knockout mice in which a particular gene is inactivated (gene knockout) can be produced in the laboratory by using embryonic stem cells (Fig. 24.3).
Embryonic stem cells (ES cells) are present in the early blastocyst stage of mammalian embryos. The layer of cells lining the blastocyst, called trophoectoderm, contains on its inner surface a cluster of cells referred to as the inner cell mass that projects into a cavity called blastocoel. The inner cell mass contains the embryonic stem cells which differentiate into all the cell types present in all tissues of the adult animal.
ES cells can be isolated from the blastocyst and established in culture where they proliferate. A DNA fragment that contains a non-functional mutant allele of the gene to be knocked out is transfected into ES cells. A gene for antibiotic resistance is also incorporated in the fragment to allow for selection of cells that have incorporated the inactivated gene into their genome.
Among the ES cells that take up the DNA fragment, about one cell in 104 cells undergoes the process of homologous recombination by which the transfecting DNA sequence replaces homologous DNA. In this way, ES cells that have acquired the transfecting gene become heterozygous for this particular gene.
That is because the homologous sequence in identical location in the other chromosome is carrying the normal allele of this gene. ES cells are grown on medium and those with the gene of interest are selected on the basis of antibiotic resistance.
The selected ES cells (donor cells) are then injected into the blastcoel of a recipient mouse embryo. The recipient embryo is implanted into a female mouse, pretreated hormonally to carry the embryo to term. The injected ES cells in the surrogate mother join the embryo’s own inner cell mass, and as the embryo develops, these ES cells become part of some embryonic tissues including the reproductive gonads.
The progeny mice are found to be chimaeric because they display characteristics of both donor and recipient. The word chimaera is derived from the mythological creature Chimaera with the head of a lion, body of a goat and tail of a serpent. To find out whether or not the germ cells contain the knockout gene, the chimeric mice are mated to normal mice.
If the germ cells contain the knockout gene, the progeny mice will be heterozygous for this gene. Two heterozygous mice are then mated to one another to obtain some mice that are homozygotes, according to mendelian inheritance, and are called knockout mice.