Biomedical Engineering Reference
In-Depth Information
mice. Chimeric mice are mice that contain cells of more than one geno-
type. The name chimera has its origins in Greek mythology and is taken
from the Greek word meaning “she-goat”. This mystical creature was
the offspring of Echinda and Typhon and had the body of a goat, the
head of a lion, and the tail of a serpent. Such chimeric creatures have
often been represented in mythology. Other examples include centaurs,
half horse and half man, the sphinx, with a human head on a lion's body,
and the minotaur, which is half bull and half man. There is also a natural
form of a chimera that exists in nature, and many cattle farmers will
be familiar with it. Twin cattle share a placental membrane, and con-
sequently there is an exchange of blood between the twins. When the
twins are male and female, the female, called a “freemartin” is usually
infertile: even though the animal is genetically female, it will have many
male characteristics. But, I digress.
In constructing chimeric mice using ES cells, combinations of ES cells
and donor blastocysts are used which derive from mice that differ in coat
color. As a result, chimeric offspring are obvious since they contain coats
that are not uniform in color, but are made up of the two different donor
cells. The chimeric mice are then bred; if the ES cells have contributed
to germ cells (sperm or egg), then offspring containing the modified
gene will be obtained. These can be crossed to generate homozygous
knockout mice.
Knockin mice
Knockin mice are generated in much the same way. The only real
difference is that instead of being designed to eliminate an endogenous
gene, the targeting construct is made so that the endogenous gene will
be altered. For example, coding sequences can be mutated in order to
generate a mouse model of a human mutation, or regulatory sequences
can be changed in order to alter the expression of a gene. Alternatively,
the endogenous mouse gene can be replaced by its human counterpart.
Floxed genes in knockout technology
Although knockouts were originally generated using genes in which
an exon(s) had been replaced by the neo gene, it became clear that
this gene, and its accompanying promoter, could introduce artifacts of
its own. Consequently, a method to eliminate the neo marker was devel-
oped. This takes advantage of a P1 bacteriophage recombinase, Cre,
and its binding sites, loxP. LoxP sites are 34 bp sequences with an
8 bp core sequence (that determines directionality of the recombina-
tion event), and flanked by 13 bp palindromic sequences. When a DNA
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