Biomedical Engineering Reference
In-Depth Information
The transposition of
Drosophila
P elements appears
to be restricted to drosophiloid insects (Handler
et al.
1993), but other transposable elements are more
promiscuous. The
Ac-Ds
elements of maize have
been shown to function in a wide range of plants -
dicots as well as monocots - and have been extens-
ively used as insertional mutagens and gene tags
in heterologous species (reviewed by Sundaresan
1996). T-DNA can also be used as a tag following
transfer by
Agrobacterium tumefaciens
. T-DNA muta-
genesis and tagging has been used in
Arabidopsis
(Feldmann 1991, Krysan
et al.
1999) and has recently
been extended to other plant systems, such as rice
(Jeon
et al.
2000).
Entrapment constructs
Entrapment constructs are genetic tools that com-
bine three important principles of gene-transfer
technology: (i) the random integration of transgenes
causes insertional mutagenesis and tags the mutated
gene; (ii) randomly integrated DNA sequences are
subject to variable position effects (Box 11.1); and
(iii) reporter genes can be used to assay the activity
of regulatory elements to which they become joined
(Box 10.1). Entrapment constructs are insertional
mutagenesis vectors adapted to provide information
about the genomic region into which they integrate.
Such vectors contain a reporter gene, whose ex-
pression is activated by regulatory elements in the
surrounding DNA. When they integrate into the
genome, the pattern of reporter-gene expression
reveals the activity of nearby genes, allowing in-
vestigators to screen for appropriate or interesting
insertion events. The corresponding gene can be
cloned, because it is tagged by the insertion.
Vectors for insertional mutagenesis and tagging
The development of vectors specifically for insertional
mutagenesis and gene tagging arose directly from
the use of transposons and the recovery of serendip-
itous insertional mutants in gene-transfer experi-
ments. The transposable elements of several species
have been developed as vectors. Modifications have
been carried out in order to control the number of
insertion events and to facilitate the cloning and
analysis of tagged genes. The use of recombinant P-
element derivatives for controlled transposon muta-
genesis in
Drosophila
is a good example (reviewed by
Cooley
et al.
1988). Two fly strains are involved. One
strain contains the
mutator
, a defective P element
carrying useful marker genes, which can be mobil-
ized when provided with a source of transposase.
A second strain contains the
jumpstarter
, a
wings-
clipped
element, which provides transposase
in trans
but lacks the
cis
-acting elements required for its
own mobilization. During a controlled mutagenesis
screen, a single jumpstarter element is crossed into a
mutator-containing strain, whereupon transposition
occurs. In subsequent generations the mutator is
stabilized when the chromosome carrying the mutator
element segregates from the chromosome bearing
the jumpstarter element. The inclusion of a marker
gene in the mutator P element allows screening or
selection for transformed flies and facilitates subse-
quent manipulation of the modified locus. The inclu-
sion of a bacterial selectable-marker gene and an
E. coli
origin of replication allows recovery of the
tagged gene by plasmid rescue, as discussed above.
Enhancer traps in
Drosophila
The prototype entrapment construct was the
Droso-
phila
enhancer trap, which originated as an import-
ant application of P-element vectors (p. 219). The
method employs a
lacZ
reporter construct, in which
the reporter gene is transcribed from a minimal
promoter, such as a TATA box. Expression from the
promoter is weak, because the promoter lacks an
enhancer to stimulate its transcriptional activity. P-
element-mediated transposition is used to transpose
the construct into many different genomic positions
in separate fly lines. In some flies, by chance, the
construct is transposed to a position where it comes
under the influence of an enhancer that activates
transcription from the weak promoter (Fig. 13.13).
It is often found in practice that, when using a histo-
chemical stain for
-galactosidase activity, the pat-
tern of expression shows cell specificity. Sometimes
the pattern of expression is remarkably refined and
detailed (O'Kane & Gehring 1987). The pattern of
lacZ
expression is assumed to reflect the cell-type
specificity of the enhancer. Presumably, an endogen-
ous gene located within range of the enhancer's
effect has the same pattern of expression as the
reporter. This assumption is known to be valid in
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