Biomedical Engineering Reference
In-Depth Information
1990b). These principles included the use of vectors
containing chloroplast homology regions, allowing
targeted integration into the chloroplast genome,
and the use of the selectable marker gene
aad
(amino-
glycoside adenyltransferase), which confers resistance
to streptomycin and spectinomycin (Zoubenko
et al.
1994). Efficient chloroplast transformation has
been achieved both through particle bombardment
(e.g. Staub & Maliga 1992) and polyethylene glycol
(PEG)-mediated transformation (Golds
et al.
1993,
Koop
et al.
1996). The use of a combined selectable -
screenable marker (
aad
linked to the gene for green
fluorescent protein) allows the tracking of transplas-
tomic sectors of plant tissue prior to chlorophyll
synthesis, so that transformed plants can be rapidly
identified (Khan & Maliga 1999).
manner, it is possible to generate transiently trans-
formed cell lines or transgenic plants carrying an
integrated recombinant viral genome. In the case of
RNA viruses, transcription of an integrated cDNA
copy of the genome yields replication-competent
viral RNA, which is amplified episomally, facilit-
ating high-level transgene expression. Transgenic
plants are persistently infected by the virus and
can produce large amounts of recombinant protein.
In the case of DNA viruses,
Agrobacterium
-mediated
transient or stable transformation with T-DNA
containing a partially duplicated viral genome can
lead to the 'escape' of intact genomes, which then
replicate episomally. The latter process, known as
'agroinfection' or 'agroinoculation', provides a very
sensitive assay for gene transfer.
As well as their use for the expression of whole
foreign proteins, certain plant viruses have recently
been developed to present short peptides on their
surfaces, similar to the phage-display technology
discussed on p. 136. Epitope-display systems based
on cowpea mosaic virus, potato virus X and tomato
bushy stunt virus have been developed as a potential
source of vaccines, particularly against animal viruses
(reviewed by Johnson
et al.
1997, Lomonossoff &
Hamilton 1999).
Plant viruses as vectors
As an alternative to stable transformation using
Agrobacterium
or direct DNA transfer, plant viruses
can be employed as gene-transfer and expression
vectors. There are several advantages to the use
of viruses. First, viruses are able to adsorb to and
introduce their nucleic acid into intact plant cells.
However, for many viruses, naked DNA or RNA is
also infectious, allowing recombinant vectors to be
introduced directly into plants by methods such as
leaf rubbing. Secondly, infected cells yield large
amounts of virus, so recombinant viral vectors have
the potential for high-level transgene expression.
Thirdly, viral infections are often systemic. The virus
spreads throughout the plant, allowing transgene
expression in all cells. Fourthly, viral infections are
rapid, so large amounts of recombinant protein can
be produced in a few weeks. Finally, all known plant
viruses replicate episomally; therefore the trans-
genes they carry are not subject to the position
effects that often influence the expression of integ-
rated transgenes (Box 11.1). Since plant viruses
neither integrate into nor pass through the germ
line, plants cannot be stably transformed by viral
infection and transgenic lines cannot be generated.
However, this limitation can also be advantageous
in terms of containment.
A complete copy of a viral genome can also be
introduced into isolated plant cells or whole plants
by
Agrobacterium
or direct DNA transfer. In this
DNA viruses as expression vectors
The vast majority of plant viruses have RNA genomes.
However, the two groups of DNA viruses that are
known to infect plants - the caulimoviruses and the
geminiviruses - were the first to be developed as vec-
tors, because of the ease with which their small, DNA
genomes could be manipulated in plasmid vectors.
Cauliflower mosaic virus
The type member of the caulimoviruses is CaMV.
The 8 kb double-stranded DNA (dsDNA) genome
of several isolates has been completely sequenced,
revealing an unusual structure characterized by the
presence of three discontinuities in the duplex. A
map of the CaMV genome is shown in Fig. 12.15.
There are eight tightly packed genes, expressed as
two major transcripts: the 35S RNA (which essen-
tially represents the entire genome) and the 19S
RNA (which contains the coding region for gene VI).
