modifying recombinant genes | expression in plants | risks and safety
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modifying
recombinant
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how do they prepare
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how do they get the
genes into the plant?
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are recombinant proteins
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Agrobacterium-Mediated Transformation
So how do they get the gene into the plant? This first
system has been the more popular of plant
transformation techniques in recent years. In this
particular system, the Agrobacterium tumefaciens
bacterium, which is a soil pathogen, acts as the vector
for the antibody- or cytokine-producing gene:
biotechnology
essentially
takes
advantage
of
Agrobacterium's natural gene insertion behaviour to
insert the transgene, rather than its own genetic
material, into the plant genome. After this insertion, the
plant then happily expresses the transgene to produce
the antibody or cytokine of interest as if it were the
plant's own gene. The details of this method are
described below.
Agrobacterium tumefaciens usually causes tumours or
crown galls in plants by injecting genes from a tumourinducing (Ti) plasmid into the plant's nuclear genome[1].
The wildtype Ti plasmid most importantly contains a
region called the T-DNA region (for transferred DNA),
which encodes genes involved in auxin and cytokinin
(plant hormone) synthesis. Once integrated into the
plant nucleus, these gene products will induce
deregulated growth and tumours in the plant. The TDNA also includes genes for the synthesis of opines,
compounds rather specific to the support of
Agrobacterium's growth (few other bacteria metabolise
opines)[1]. Also on the Ti plasmid are vir (virulence)
genes which will recognise plant signals and work to
inject the T-DNA into the plant. The encoded Vir
proteins slice the bacterial DNA at the T-DNA border
repeat sequences and physically relocate the T-DNA as
a single-stranded T-DNA molecule (T-strand) to the
plant's nucleus, where the T-strand integrates into the
genome, probably by taking advantage of cellular DNA
repair mechanisms[1,2].
In introducing a recombinant gene, the transgene will
be substituted for Agrobacterium's usual T-DNA
sequences (i.e. the auxin, cytokinin, and opine
synthesis genes) so that no tumour forms. However,
the border repeat sequences are preserved so that the
Vir proteins can still recognise the T-DNA and move it.
The vir genes themselves are also untouched, so that
the mechanism of transfer is not disturbed. Often, a
selectable marker is also inserted into the plasmid to
allow detection of sucessful transformation events:
transgenic plants can be easily recognized by their
persistent
grow
in
selective
media
(e.g.
hygromycin)[1,3]. See the diagram below for a full
schematic.
This
method
is
highly
useful
because
Agrobacterium tumefaciens can infect virtually
any plant, and it is fairly cheap. For example,
tobacco, pea, rice, wheat and corn plant are favourite
systems [4]. Unfortunately, only genomic integration is
possible, so any episomal DNA inserts are out of the
question. As well, transformation is only transient[3].
Gene Gun Transfer
This
method,
also
known
as
particle
bombardment, seems to be slightly less popular,
but is the only other one currently available to
researchers.
In this
method, the
created
transgene cassette is placed in a vector (plasmid)
and many copies of it are coated onto a
microscopic gold-coated particle. This particle is
accelerated into the plant cell nucleus, where the
plasmids will deattach from the particle and
hopefully integrate into the plant genome.
Transformation by particle bombardment is by no
means permanent either, and like Agrobacterium
transformation, also prone to superfluous DNA
(unnecessary, i.e. vector DNA or the selectable marker)
transfer[3,5].
→ next
references
1) Tzfira T, Citovsky V. Agrobacterium-mediated genetic
transformation of plants: biology and biotechnology.
Curr Opin Biotechnol 2006, Feb 2; [Epub ahead of
print].
2)
Gelvin,
SB.
Agrobacterium-mediated
plant
transformation: the biology behind the "gene-jockeying"
tool. Microbiol Mol Biol Rev 2003, 67: 16-37.
3) Ma JK, Drake PM, Christou P. The production of
recombinant pharmaceutical proteins in plants. Nat Rev
Genet
2003,
4:
794-805.
4) Schillberg S, Twyman RM, Fischer R. Opportunities
for recombinant antigen and antibody expression in
transgenic plants--technology assessment. Vaccine
2004,
23:
1764-1769.
5) Department of Soil and Crop Sciences at Colorado
State University, 1999-2004. How to make Transgenic
Plants:
Animation
Demo.
http://cls.casa.colostate.edu/TransgenicCrops/
animation.html , accessed Mar 20, 2006.
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