THE DERIVATION AND MANIPULATION OF EXPERIMENTAL ANIMALS IN BIOMEDICAL SCIENCES - A Guide to Methods in the Biomedical Sciences - page 109

Figure 11. Productionofatargeted transgenic by homologous recombination
confers resistance to a drug that will otherwise kill the ES cells, such
as the neomycin resistance gene (neo) which is commonly used for
this purpose. Because homologous recombination is a rare event, the
targeting construct often contains another selectable marker outside of
the flanking regions of homology (Figure 11). This selectable marker
is one which will kill ES cells that incorporate it into the DNA. A gene
encoding a toxin, such as diphtheria toxin, can be used. Alternatively,
a gene that confers sensitivity to a drug is often employed for negative
selection. One example is the herpes simplex virus thymidine kinase
(TK) gene, which confers sensitivity to the drug gancyclovir. By using a
double selection system, cells that randomly incorporate the targeting
vector as a transgene are eliminated, while cells that have incorporated
it by homologous recombination are positively selected. Consequently,
ES cells to be used for the next step in the process are rapidly identified.
The targeting construct is introduced into the ES cell line by elec-
troporation, and the cells' enzymatic machinery does the magic. In ES
cells that survive selection, the change in the endogenous gene is con-
firmed by Southern blot analysis. The modified ES cells are injected
into blastocysts, an early stage in embryogenesis characterized by a
sphere of cells enclosing an inner mass of cells in a fluid-filled cavity,
which have been harvested from donor female mice. Several injected
blastocysts are then implanted into the uterine horn of pseudopregnant
recipient mice. If these survive to full term, they give rise to “chimeric”