Biomedical Engineering Reference
In-Depth Information
diphtheria toxin), leading to cell death and thus
ablation of specific cell lineages in the fly. This has
enormous potential, e.g. in studies of the develop-
ment of the nervous system (O'Kane & Moffat 1992).
In order to facilitate the use of enhancer trapping
as a general method for driving cell-specific expres-
sion, a modified strategy has been developed. This
depends upon the ability of the yeast transcription
factor GAL4 to activate transgenes containing its
recognition site in the heterologous environment
of the fly. The enhancer-trap principle shown in
Fig. 13.13 is modified so that the
lacZ
reporter gene is
replaced by the coding region for GAL4 (Fig. 13.14).
In such flies, GAL4 is now expressed in the pattern
dictated by a local enhancer, although this cannot
be seen. The pattern of GAL4 expression can be
revealed by crossing the enhancer-trap line to flies
carrying a reporter transgene in which the
lacZ
gene
is coupled to a promoter containing GAL4-binding
sites. The beauty of this system is that a bank of fly
stocks with different trapped enhancers can be built
up, each with a defined pattern of GAL4 expression.
Once the patterns of GAL4 expression are known,
crosses can be performed to introduce chromosomes
containing constructs in which any desired gene is
coupled to a GAL4-dependent promoter, giving a
particular cell-specific pattern of expression.
Enhancer action
Randomly transposed
P-element having a weak
promoter driving
lacZ
transcription
lac
Z
Genome
Enhancer
Promoter
Fig. 13.13
Enhancer trapping. A reporter construct consists
of
lacZ
linked to a weak promoter, which requires an enhancer
for significant transcriptional activity. P-element-mediated
transposition is used to insert the construct at random sites
in the fly genome. When the promoter is inserted within
the active range of an enhancer, expression of
lacZ
can
be detected.
some cases, but the reporter activity does not always
exactly match that of an endogenous gene.
Since enhancers are often found a considerable
distance from the gene they activate, the enhancer
trap cannot be used to directly clone genes by
tagging, although it can be used as the basis for a
chromosome walk to identify the gene of interest.
Enhancer-trap lines have been used to identify and
clone novel
Drosophila
genes, but the cell-type-specific
expression that is revealed can also be harnessed in
other ways. For example, instead of driving expres-
sion of a
lacZ
reporter, the principle could be applied
to the expression of a toxic gene (such as ricin or
Enhancer trap line
UAS-
lac
Z line
E
P
GAL 4
UAS
Lac
Z
Fig. 13.14
Second-generation
enhancer trap, in which the yeast gene
for the GAL4 transcription factor is
activated by the enhancer. This can
be crossed to a responder line in which
lacZ
is driven by a GAL4-dependent
promoter.
E
P
GAL 4
GAL 4
UAS
lac
Z
