Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

STEM CELLS AND Renewal IN EPITHELIAL TISSUES
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stem-cell-specific
promoter
GFP
CreERT2
ubiquitous
promoter
tamoxifen
added
GFP marks
stem cell
LacZ
inactive
recombinase
activated recombinase
removes blocking
sequence
blocking
sequence
LacZ now expressed in
this cell and all its progeny
Figure 22–5 Clonal analysis using a genetic marker. A modern method for tracking cell lineage
uses transgenic animals containing two transgenes, which together drive expression of a readily
detected and heritable marker protein in a small subset of stem cells. The first transgene (top)
carries two adjacent protein-coding sequences, GFP and CreERT2, both expressed under the
control of the Lgr promoter that is active only in stem cells and not in their differentiated progeny.
GFP encodes green fluorescent protein (see MBoC6 Chapter n22.101/22.05
9), which is used here simply to confirm
expression in the entire stem-cell population. The CreERT2 gene encodes a chimeric form of the
Cre recombinase called CreERT, which consists of Cre recombinase linked to the estrogen receptor
protein; this enzyme becomes active as a recombinase only when it binds the artificial estrogen
analog tamoxifen.
The second transgene (bottom) carries a marker gene, LacZ, under the control of a promoter
that is active in all cells. The LacZ gene encodes β-galactosidase, an enzyme that can be detected
histochemically in tissues (see Figure 7–28). However, LacZ expression in the transgene shown
here is prevented by a blocking sequence (red) that is flanked by LoxP sites (pink; see Figure
5–66). When tamoxifen is provided, CreERT becomes active––leading to a recombination event
that removes the blocking DNA sequence (and leaves one LoxP site behind). As a result, the LacZ
marker is expressed. Because this change is heritable, the marker continues to be expressed in
all cells descended from those in which a recombination event has occurred. With a low dose of
the inducer molecule tamoxifen, it is possible to activate the marker at random in just a few widely
spaced cells, which, in the course of time, give rise to widely separated and easily distinguished
clones of progeny (see Figure 22–6).
is descended from whom, or, equivalently, what progeny will be produced from
any given cell. This can best be done using a heritable marker that can be activated
in an individual cell, thus allowing the identification of the clone of progeny
descended from that cell. A modern method uses transgenic animals to create
a visible genetic mark in just a few widely spaced cells, which, in the course of
time, give rise to widely separated and easily distinguished clones of progeny, as
explained in Figure 22–5.
A search among genes that are strongly upregulated in response to Wnt signaling
revealed one, called Lgr5, that is expressed in gut stem cells specifically.
The technique described in Figure 22–5 can be used to create a genetic mark in a
random subset of Lgr5-expressing cells—a mark that is inherited by the progeny
of each cell. These Lgr5 cells divide with a cycle time of about 24 hours, and within
a few days marked clones are seen extending from the crypt bases up along the
sides of the villi. After as long as 60 days or more, many of these clones still persist,
retaining one or more members at the crypt base and extending all the way up to
the tips of the villi (Figure 22–6). Moreover, each single clone typically contains
all the major differentiated gut cell types—absorptive, goblet, Paneth, and enteroendocrine—in
their normal proportions. The Lgr5-expressing cells, therefore, are
true stem cells that are multipotent—that is, able to generate a diverse set of differentiated
cell types.