Biomedical Engineering Reference
In-Depth Information
approach has been exploited very widely for examin-
ing the developmental effects resulting from the
overexpression of normal or altered gene products
(reviewed by Vize & Melton 1991).
DNA can be introduced into
Xenopus
embryos
in the same manner. However, unlike the situation
in mammals, where the injected DNA integrates
rapidly into the genome, exogenous DNA in
Xenopus
persists episomally and undergoes extensive replica-
tion (Endean & Smithies 1989). Bendig and Williams
(1983) provide a typical example of this process.
They injected a recombinant plasmid carrying
Xenopus
globin genes into the egg and showed that the
amount of plasmid DNA increased 50- to 100-fold
by the gastrula stage. In later development, the
amount of DNA per embryo decreased, and most
of the persisting DNA co-migrated with high-
molecular-weight chromosomal DNA. This difference
between mammals and amphibians probably re-
flects their distinct modes of early development. In
mammals, cleavage divisions are slow and asyn-
chronous. Gene expression occurs throughout early
development and supplies the embryo with the
proteins it requires at a steady rate. Conversely,
there is no transcription in the early
Xenopus
embryo
and yet the cleavage divisions are rapid and syn-
chronous. DNA replication relies on stored maternal
gene products, so there is a stockpile of chromatin
assembly proteins and replication enzymes. Exogen-
ous DNA injected into
Xenopus
eggs is therefore
assembled immediately into chromatin and under-
goes replication in tune with the rapid DNA synthesis
already occurring in the nucleus (Leno & Laskey
1991). Etkin
et al
. (1987) have analysed the replica-
tion of a variety of DNAs injected into
Xenopus
embryos. It was found that various plasmids
increase to different extents. This was not simply
related to the size of the plasmid, but also reflected
the presence of specific sequences that inhibited
replication. Replication has also been found to
depend upon the conformation and number of
molecules injected (Marini
et al
. 1989).
Tissue
Extraction
Affinity chromatography
Size
fractionation
Poly(A)
+
RNA
Microinjection
Microinjection
Xenopus
oocyte
Poly(A)
+
RNA
Assay for
functional
expression
cDNA library
Synthesis
In vitro
transcription
and capping
Fig. 11.8
Strategy for functional expression cloning, using
Xenopus
oocytes as a heterologous expression system.
stomach mRNA, using a vector in which the cDNA
was flanked by a promoter for the SP6 or T7 RNA
polymerase. This allowed
in vitro
synthesis of mRNA
from the mixture of cloned cDNAs in the library.
The receptor clone was identified by testing for
receptor expression following injection of synthetic
mRNA into the oocyte cytoplasm. Repeated sub-
division of the mixture of cDNAs in the library led
to the isolation of a single cloned cDNA. The strat-
egy described above can only be applied to cloning
single-subunit proteins, not proteins composed of
different subunits or proteins whose function in
oocytes requires more than one foreign polypeptide.
This limitation was overcome by Lubbert
et al
.
(1987), who used a hybrid depletion procedure to
clone a serotonin-receptor cDNA.
A prerequisite for using the oocyte in functional
expression cloning is a knowledge of the oocyte's
own ion channels, carriers and receptors. Endogen-
ous activity may mask or interfere with the sought-
after function (for a review, see Goldin 1991).
Transient gene expression in
Xenopus
embryos
Messenger RNA, synthesized and capped
in vitro
,
can be microinjected into dejellied
Xenopus
embryos
at the one- or two-cell stage. The mRNA is distri-
buted among the descendants of the injected cells
and is expressed during early development. This
Transgenic
Xenopus
DNA injected into early
Xenopus
embryos is ex-
pressed in a mosaic fashion during development,
regardless of the promoter used, which limits the use
