Advances in transgenic technology - Principles of Gene Manipulation - page 273

SA IRES lac Z/neo polyA P pur SD

Fig. 13.16 A highly versatile gene-trap vector designed for high-throughput screening. See text for details.

et al. 2000). Such a library of clones can be grown,
stored frozen in microtitre plates and systematically
analysed to build up a molecular library of flanking
sequences. In order to characterize the expression
patterns of the trapped genes, it is possible to under-
take large-scale screens of mouse embryos or par-
ticular tissues of adult mice produced from the ES
cells, but prescreening is also possible by exploiting
the ability of ES cells to differentiate along different
pathways in specific culture media (e.g. Reddy et al.
1992, Baker et al. 1997, Yang et al. 1997). It is also
possible to identify ES cells in which particular
functional subsets of genes are trapped, e.g. secreted
proteins (see below). An elegant gene-trap vector
suitable for high-throughput screening has been
designed by Zambrowicz et al. (1998) (Fig. 13.16).
There are two expression cassettes in this vector:
the

biased in favour of genes that are actively expressed
in ES cells.
Recently, a Xenopus gene trap has also been
developed, based on the transgenesis procedure of
Kroll and Amaya (1996) (p. 216). In this system, the
reporter gene for green fluorescent protein is used
(Box 13.1), allowing reporter expression patterns
to be analysed in living tadpoles (Bronchain et al.
1999). This system could become a powerful tool for
functional genomics in Xenopus because hundreds
or thousands of embryos can be generated in a single
day and screened for gene-trap events in real time.

Function-specific trapping

The design of gene-trap constructs can be modified
to select for particular classes of genes. For example,
Skarnes et al. (1995) describe a construct in which
the

-geo hybrid marker downstream of a splice
acceptor and internal ribosome entry site, and the
selectable marker pur , conferring resistance to puro-
mycin, under the control of a constitutive promoter
but lacking a polyadenylation signal, upstream of
a splice donor site. Using this vector, expression of
the reporter is dependent on the formation of a
transcriptional fusion with the trapped gene, but,
since the selectable marker is driven by its own
promoter but dependent on the structure of the gene
for correct processing, selection for insertions is not

β

-geo marker is expressed as a fusion to the
transmembrane domain of the CD4 type I protein. If
this inserts into a gene encoding a secreted product,
the resulting fusion protein contains a signal peptide
and is inserted into the membrane of the ER in the
correct orientation to maintain

β

-galactosidase
activity. However, if the construct inserts into a
different type of gene, the fusion product is inserted
into the ER membrane in the opposite orientation
and

β

β -galactosidase activity is lost.