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Gene transfer to plants
The
Pharma-Planta consortium uses several different technologies for getting genes
into plants. To transform tobacco we use Agrobacterium
tumefaciens. To transform maize we use particle bombardment. To transform
plastids of tobacco, tomato and lettuce, we use particle bombardment or
PEG-mediated protoplast transformation.
The
figure below shows the general Agrobacterium method for DNA transfer to tobacco
plants.
The
figure below shows some tobacco protoplasts, cells denuded of their cell walls,
which are used for plastid transformation.
Gene expression in plants
Our
pharmaceutical genes are expressed under the control of promoters that function
in specific plant cells and species. In tobacco, we use a ubiquitous promoter which
drives expression in leaves and all other tissues. In maize, we use
seed-specific promoters to make the pharmaceutical protein accumulate in the
kernels. For plastid transformation, the gene inserts in line with an
endogenous plastid gene and is expressed under that gene’s promoter.
The
figure below shows a protein blot, which proves that the pharmaceutical protein
(the HIV antigen p24) is present in the tobacco tissue.
Identity preservation
To
make sure we can track transgenic plant material, all our transgenic
pharmaceutical plants are cotransformed with the visible marker DsRed. This is
a fluorescent protein that makes the plant tissue glow red under an appropriate
light source.
The
figure below shows second generation maize cobs with a mixture of transgenic
(orange) and non-transgenic (green) seeds.
.