Essential Cell Biology 5th edition

356 CHAPTER 10 Analyzing the Structure and Function of Genes
replaced by the altered one. In this way, the function of the mutant protein
can be analyzed in the absence of the normal protein. A common way
of doing this in mice makes use of cultured mouse embryonic stem (ES)
cells (discussed in Chapter 20). These cells are first subjected to targeted
gene replacement before being transplanted into a developing embryo to
produce a mutant mouse, as illustrated in Figure 10–28.
Using a similar strategy, the activities of both copies of a gene can be
eliminated entirely, creating a “gene knockout.” To do this, one can
either introduce an inactive, mutant version of the gene into cultured ES
cells or delete the gene altogether. The ability to use ES cells to produce
such “knockout mice” revolutionized the study of gene function, and the
(A)
ES cells growing
in culture
(B)
pregnant mouse
altered version
of target gene
constructed by
genetic
engineering
INTRODUCE A DNA
FRAGMENT CONTAINING
ALTERED GENE INTO
MANY CELLS
LET EACH ES CELL
PROLIFERATE TO
FORM A COLONY
INJECT ALTERED
ES CELLS
INTO EARLY
EMBRYO
ISOLATE EARLY
EMBRYO
Figure 10–28 Targeted gene replacement
in mice utilizes embryonic stem (ES)
cells. (A) First, an altered version of the
gene is introduced into cultured ES cells.
In a few rare ES cells, the altered gene will
replace the corresponding normal gene
through homologous recombination (as
described in Chapter 6, pp. 220−222 and
Figure 6−31). Although the procedure is
often laborious, these rare cells can be
identified and cultured to produce many
descendants, each of which carries an
altered gene in place of one of its two
normal corresponding genes. (B) Next, the
altered ES cells are injected into a very early
mouse embryo; the cells are incorporated
into the growing embryo, which then
develops into a mouse that contains some
somatic cells (colored orange) that carry the
altered gene. Some of these mice may also
have germ-line cells that contain the altered
gene; when bred with a normal mouse,
some of the progeny of these mice will
contain the altered gene in all of their cells.
Such a mouse is called a “knock-in” mouse.
If two such mice are bred, one can obtain
progeny that contain two copies of the
altered gene—one on each chromosome—
in all of their cells.
TAKE CELLS FROM THE
RARE COLONY IN WHICH
THE DNA FRAGMENT
HAS REPLACED ONE
COPY OF THE
NORMAL GENE
ES cells with one copy of target
gene replaced by altered gene
some of these
offspring have
germ-line cells
containing altered gene
the offspring will
include males and
females with one copy
of target gene
altered in all cells
EARLY EMBRYO FORMED
PARTLY FROM ALTERED
ES CELLS
INTRODUCE EARLY
EMBRYO INTO
PSEUDOPREGNANT
MOUSE
BIRTH
MATE WITH
NORMAL MOUSE
MATING
TRANSGENIC MOUSE
IN WHICH BOTH COPIES OF
TARGET GENE ARE ALTERED