Questions'''agree: green disagree: red

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Questions...agree greendisagree red

• In order for a transgene to be expressed in
  plants, you need to first clone it into an
  expression vector.
• The expression vector contains a promoter that
  directs the expression of the transgene.
• Dr. Labuznik is heard boasting I cloned the
  luciferase gene from the fire fly and transformed
  it into tobacco. Now my plants are glowing in the
  dark. He needs to be reprimanded.
• The main advantage of Agrobacterium mediated
  transformation is that it can be used with any
  plant.
• Transient expression is heritable
• The efficiency of regeneration of transgenic
  plants from transformed cells is aided by the use
  of a selectable marker.

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The gene gun
The Helios Gene Gun is a new way for in vivo
transformation of cells or organisms (i.e. gene
therapy and genetic immunization (DNA
vaccination)). This gun uses Biolistic particle
bombardment where DNA- or RNA-coated gold
particles are loaded into the gun and you pull
the trigger. A low pressure helium pulse delivers
the coated gold particles into virtually any
target cell or tissue. The particles carry the
DNA so that you do not have to remove cells from
tissue in order to transform the cells.
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• Genetic Engineering
• So, we now know how to clone a gene and we know
  how to make transgenic plants. We have already
  looked at some uses of transgenic plants in basic
  science (promoter bashing, T-DNA insertions.
  Lets look at some applications of genetic
  engineering and some of the concerns this has
  raised.
• There are several approaches to genetic
  engineering.
• knocking out existing genes. This is in
  essence similar to the creation of mutations,
  except that you know precisely which gene you
  inactivate. The resulting phenotype resembles
  that of a mutant, but unlike mutations, which are
  typically recessive, the gene knock-out based on
  genetic engineering is dominant. There are two
  ways to knock out a gene through the use of
  antisense strategies or the use of
  co-suppression. Details to follow.
• Enhanced expression of existing genes. The goal
  is to generate more of a particular enzyme to
  remove bottlenecks in a metabolic process. This
  is typically achieved by introducing an
  additional copy of a gene of interest into the
  genome, under control of a strong promoter. Has
  risk of co-suppression!
• Introduction of foreign genes. This refers to
  the introduction of genes that were not part of
  the genome. In this case the boundaries between
  kingdoms can be crossed. We can for example
  introduce a fish gene in a tree, or a pig gene in
  a bacteria. Two reasons for doing this are
• To create a more efficient process or a process
  that results in a product with enhanced quality.
  An example is the introduction of bacterial
  starch synthase genes in potato tuber.
• To create entirely new characteristics. An
  example is the introduction of herbicide
  resistance in plants, or the creation of oranges
  that can tolerate frost because of an anti-freeze
  protein from fish.

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Modifying existing metabolic processes In order
to be successful at changing a metabolic process
through genetic engineering it is important to
understand the process. This means knowing which
genes are involved, what proteins they encode,
and what the kinetic properties of enzymes are.
For example, it does not make any sense to
generate more of an enzyme that is already
abundant (i.e. not rate-limiting). It is also
important to think about possible side effects of
changing steps in a pathway. This is an area
where basic biology plays an important role.
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Antisense strategies to down-regulate genes The
idea was simple you clone the gene you want to
knock out in an expression vector, but you put it
in backwards, i.e. the 3 end of the gene is
closest to the promoter. The non-coding strand
will get transcribed and produce an antisense
transcript. Originally it was thought that
this antisense transcript would hybridize with
the sense transcript from the native gene and
therefore prevent the native transcript from
being translated. Current thinking, however,
suggests that another mechanism is responsible
for the antisense effect. This mechanism is
similar to that of co-suppression.
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Co-suppression This refers to the inactivation
of genes when transgenes are present. Both the
transgenes themselves and the native genes can
become silenced, so that you end up with the
phenotype of a mutant or a knock-out. The
mechanism behind co-suppression is just now being
elucidated. The gene silencing can happen at the
level of transcription and at the
post-transcriptional level. The latter is
referred to at PTGS or post-transcriptional gene
silencing and has been studied in more detail. A
similar mechanism is known as RNAi (for RNA
interference in the nematode C. elegans). In
order for the gene to be silenced, transcription
is required. Current models suggest that an RNA
dependent RNA polymerase generates a cRNA strand
from aberrant RNA templates (either because of
mutations or because they have not been processed
normally, as in the case of transgenes). This
then results in the formation of a dsRNA
molecule. The dsRNA molecule is then cleaved into
small fragments of 21-25 nt (both orientations).
These small RNAs are incorporated into a
ribonuclease and act as guides for
sequence-specific degradation homologous RNAs.
RNA-directed DNA methylation is another
mechanism that is likely to play a role in gene
silencing. In this case specific DNA sequences
are methylated (and thus become transcriptionally
inactive) when an RNA that shows homology to the
DNA is present. The precise mechanism is yet
unknown. RNA-based silencing mechanisms are
thought to have evolved from a defense response
against viruses with RNA genomes.
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Position effects This is a term that refers to
the fact that there can be quite a bit of
variation in the effect of a transgene when
independent transformants are compared. For
example, when 20 transformants are compared, some
may not show any effects, others mild effects and
some severe effects. And yet they were all
transformed with the same construct.. An
explanation for this is that the insertion of the
transgene is considered more or less random,
particularly for the particle bombardment based
method. The transgene may integrate in an area
of the genome that is not transcriptionally
active (heterochromatin), or a region of the
genome that is heavily methylated. In other
cases the transgene may integrate in an area that
is transcriptionally active so that it is being
transcribed at a higher incidence. Position
effects thus make it hard to interpret your data,
because there is no single effect to report. What
researchers often end up doing is pick the one or
two plants with the most severe phenotype, and do
detailed studies with those. The risk is that
the insertion of the transgene has knocked out an
existing gene and that part of the phenotype can
be attributed to that. One way of reducing
position effects is to place the transgene (and
its promoter and terminator) between MAR
sequences. Remember those? Matrix attachment
regions! So the idea is to create a
transcriptionally active loop that harbors the
transgene and is more or less insulated from the
adjoining DNA and its transcriptional state. It
helps, yet many constructs used in transformation
do not include these.
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• Genetic Engineering
• Applications of genetic engineering include the
  introduction of new (foreign) genes in a
  species. This has the potential to cause
  substantial changes in the very nature of the
  organism. Some of these changes can be highly
  beneficial, at least to the producer or consumer.
  Some examples
• Golden rice rice that contains pro-vitamin A
  has significantly higher nutritional value
• Poplar trees in which the lignin composition has
  been altered so that paper production is less
  polluting
• Crop plants that have a gene that kills pathogens
  when the plant is attacked.
• Crop plants that are herbicide resistant.
• The whole issue of genetic engineering raises
  ethical questions.
• -What defines a species?
• -Are we tinkering with nature?
• -Do we really know what we are doing when we
  introduce these genes?
• -Are we going to be minions of the big
  agribusinesses?
• -Is there a trend towards monoculture and its
  associated risks?
• -Horizontal gene transfer, particularly of
  antibiotic resistance genes?
• ..