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2-Plants: New study questions reliable drug production in GM plants

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TITLE:  Gene silencing hinders bioreactor potential
SOURCE: ISB News Report, by P. Janaki Krishna
DATE:   July 6, 1999

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Since the establishment of plant genetic transformation methods,
scientists have been evaluating plant systems as possible
bioreactors for antibodies. However, the development of
heterologous protein expression systems, especially for the
production of complex proteins such as antibodies, has been
difficult. Refinements in genetic transformation technique have
permitted scientists to target protein expression and
accumulation to specific storage organs (e.g. seeds, tubers), but
in order to use plants effectively as bioreactors to produce
antibodies, it is crucial that stability of protein expression is
as carefully controlled as the yield and location.

Transgene silencing, which affects stability of protein
expression, is a common phenomenon and a major factor hindering
the potential for plants to be used as bioreactors. Though high
accumulation levels of heterologous protein are observed in young
transgenic plants, older transformants often accumulate levels up
to 100-fold lower. In addition, primary transformants have higher
accumulation levels when compared to secondary transformants.
This reduction in protein accumulation is thought to be due to
either reversible or progressive inactivation of transcriptional
genes, or both.

Recently, a team of Belgian researchers evaluated the ability of
Arabidopsis thaliana plants to produce IgG1 antibody (AB) and its
derived Fab fragments in subsequent generations of primary
transformants. Previous work had revealed successful expression
and accumulation of Immunoglobulin G and the antibody-binding
fragments, Fab in plants. The stability of AB and Fab expression
was evaluated using five different homozygous Arabidopsis lines
(kd 4, kd 13, kd 12, kd 27, and kg 49). Transgenic lines were
obtained by co-cultivating Arabidopsis with two strains of
Agrobacterium carrying light and heavy chains of T-DNA. The
stability of Fab(AB) expression was assessed at the protein level
in different 6 week-old plants of the same line, and in different
leaves of the same plant at progressive stages of plant
development. These profiles were compared with the expression
levels of Fab(AB) at the RNA level in 8-week-old plants.

Samples from each of the five different lines were examined using
Northern analysis and methylation analyses. All plant lines
showed silencing of the transgene and subsequent instability of
antibody production. However, each line expressed a different and
specific instability profile. Because expression level and
methylation were highly variable, the authors concluded that both
transcriptional and post-transcriptional gene silencing
mechanisms were operating in a chaotic manner in these plant

Three of the five lines contained either a large (kd 4 and kd 13)
or a small (kg 49) proportion of plants showing transcriptional
silencing of the transgenes. Methylation was observed only in the
promoter regions of kd 4. However, in seed stocks of kd 13/1 and
kd 13/4, antibody accumulation corresponded to methylation in
both promoter and coding sequences. In these lines, the strong
correlation between level of transgene expression and degree of
promoter methylation suggested that an interaction between
chromatin environment, which may be epigenetically modified
before or during gametogenesis, and genotype may determine the
final accumulation levels of transgene encoded proteins
exhibiting transcriptional gene silencing.

In contrast, lines kd 12 and kd 27 had expression characteristics
suggesting that a post-transcriptional gene silencing (PTGS)
mechanism was occurring that could not be attributed to
environmental factors. Differences in expression profiles between
these two lines indicated a position effect on the initiation of
gene silencing and further showed that factors other than genetic
constitution were determining methylation patterns. Consequently,
the authors proposed that epigenetic effects were responsible for
the different transgene expression profiles in all five
Arabidopsis lines.

The results of this research indicated that transgene silencing
could continue to hinder the economic exploitation of plants as
bioreactors for antibodies and other heterologous proteins.
Further studies designed to sort out the mechanisms that trigger
gene silencing are needed in order to design constructs and
vectors capable of generating transgenic lines that are not
susceptible to silencing.


De Neve M, De Buck S, De Wilde C, Van Houdt H, Strobbe I, Jacobs
A, Van Montagu M, and Depicker A. 1999. Gene silencing results in
instability of antibody production in transgenic plants.
Molecular and General Genetics 260:582 - 592.

P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise

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