Questions'''agree: green disagree: red

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Questions...agree greendisagree red

• BLAST is an algorithm optimized to find local
  sequence alignments
• The great thing about bio-informatics software is
  that you can just point-and-click and BOOM!, you
  got your answer
• Increasing the word length (w) in BLAST is going
  to reduce the number of hits I will pick up in
  the database.
• The second half of this class is really hard

2

• Transformation
• Transformation refers to the generation of
  transgenic organisms, i.e. the introduction of a
  gene of choice into the genome of an organism.
  Also referred to as genetic engineering or
  genetic modification.
• The correct expression is We transformed (fill
  in your organism) with the (fill in name) gene
• Even the most famous scientists will make the
  mistake of saying (or worse writing) (for
  example) We transformed the resistance gene
  into tobacco. This is incorrect!! A correct
  alternative to this sentence is We introduced
  the resistance gene in tobacco via
  transformation
• Why make transgenic plants?
• Improvement of crop plants by introduction of new
  genes
• Promoter studies (tobacco Rb7 gene!)
• T-DNA tagging to clone genes
• Find out the function of a gene by overexpressing
  it or by reducing its expression

3

• Making a transgenic plant requires several steps.
• You need to have your gene of interest (also
  called YFG - your favorite gene) cloned
• YFG needs to be cloned into an expression vector.
  This is a plasmid that contains a promoter and
  terminator, so that upon introduction of YFG in
  the plant, it will not just sit there.
• The actual transformation, so this is the
  introduction of YFG into the plant tissue.
  Several techniques are available.
• Regeneration of the transformed tissue growing
  a new plant from transformed cells
• Selection of transformants
• Analysis of transgene expression
• We have spent enough time talking about cloning,
  so lets assume we have a cDNA of YFG.

4

• Expression vectors (typically plasmids, but in
  some cases viruses) can be custom-made or bought
  commercially. To insert YFG into the vector you
  need to use standard lab procedures that
  typically involve the use of E. coli as a way to
  get enough vectorDNA.
• Typical plant expression vectors contain the
  following features
• E. coli origin of replication so that the plasmid
  can be manipulated in E. coli
• A bacterial antibiotic resistance gene, typically
  ampicillin. This allows for the selection of
  bacteria that contain the plasmid.
• A plant promoter, often Cauliflower Mosaic Virus
  (CaMV) 35S promoter. This is a strong promoter
  that is expressed in a lot of plant tissues. It
  is, however, not constitutive as some people
  claim! Other promoters may be more suitable,
  particularly if YFG needs to be expressed in a
  tissue-dependent manner
• A plant selectable marker. This can be a
  herbicide resistance gene, or an antibiotic
  resistance gene (often kanamycin)
• For Agrobacterium vectors there is a separate
  origin of replication and there may be a separate
  antibiotic resistance gene.
• After construction of the vector using standard
  lab techniques (restriction digests, ligation)
  the vectors are typically introduced in E. coli
  and then propagated. The plasmid is then
  harvested and is either used for direct
  transformation or is introduced in A.
  tumefaciens.

5

• Direct transformation
• The most common technique for direct
  transformation is microprojectile or particle
  bombardment. This technique is based on the use
  of a particle gun or gene gun. The expression
  vector with YFG is precipitated onto tungsten or
  gold particles which are then shot into the plant
  tissue. In most cases we will see only transient
  expression (i.e. the DNA does not integrate into
  the genome but is transcribed until it degrades).
  In a small percentage of the cells the DNA will
  integrate and we will see stable expression. The
  main drawback of this technique is that often
  multiple copies of the transgene insert. It is
  also necessary to have an in vitro regeneration
  system in place.

Rupture disk
Disk with DNA-coated particles
Pressure gauge
Stop plate
Vacuum line
Gas line
Vacuum chamber
Sample goes here
6
Agrobacterium mediated transformation Agrobacteri
um tumefaciens and Agrobacterium rhizogenes are
pathogenic soil bacteria that contain a Ti (tumor
inducing) or Ri (root inducing) plasmid. A small
piece of this plasmid, the T-DNA is transferred
from the bacteria to the plant and integrates
stably into the plant genome. The focus will
primarily be on A. tumefaciens and the
Ti-plasmid because it is more common. The T-DNA
encodes genes that stimulate cell division
(result in production of plant hormones) and the
synthesis of opines (amino acid derivatives
either nopalines or octopines). These opines are
used by the bacteria as carbon and nitrogen
source. The Ti-plasmid contains genes that help
in the break-down of these opines. The T-DNA
(transfer-DNA) is transferred to the host. The
T-DNA is flanked by a left and a right border,
consisting of 25 bp direct repeats. These
borders are recognized by endonucleases encoded
by the virD1 and virD2 genes, which are part of
the Ti-plasmid. The virD2 protein nicks the
border sequence and binds to the 5 end of the
nicked DNA. The T-DNA is thought to unwind from
the plasmid and is then transferred to the plant
as a single-stranded DNA molecule. It is coated
with the virE2 protein, which is a DNA binding
protein with a nuclear import signal that targets
the T-DNA to the plants nucleus, where it
integrates into the genome. The use of this
naturally occurring gene transfer process as a
transformation tool was made possible by the
discovery that the Ti-plasmid could be disarmed,
i.e. the tumor inducing genes on the T-DNA could
be eliminated without affecting the gene transfer
itself. So you could replace the undesirable
genes with YFG! The modern expression vectors
can replicate in both E. coli and A. tumefaciens.
So you can construct the plasmid in E. coli,
harvest large quantities of it, make sure it is
OK, and then transfer it to A. tumefaciens via
electroporation (an improvement over the
tri-parental matings that were used in the
beginning).
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The actual transformation process involves
infecting the plant tissue with A. tumefaciens
carrying the newly designed Ti-plasmid. The
general method involves the use of a leaf punch
that is incubated in a suspension of bacteria.
The cells on the edge of the punch are
transformed. These transformed cells now need to
develop into new plants through tissue culture.
The leaf disk is typically placed on a medium
that stimulates shoots (high cytokininauxin
ratio), selects for transformants (based on the
selectable marker) and kills Agrobacterium
(carbenicillin). The shoots that form are placed
on a root-inducing medium (low cytokininauxin
ratio) and the resulting plantlets are eventually
transferred to soil. This regeneration process
is very similar for tissues transformed with the
particle gun. The latest Agrobacterium-based
transformation technique in Arabidopsis is
referred to as floral dip. In this case the
tissue culture stage has been eliminated.
Instead, the Arabidopsis plant is dipped into a
suspension of Agrobacterium at the stage where it
has the maximum number of unopened floral bud
clusters. The suspension also contains a
detergent to reduce surface tension, and some
sugar. The bacteria get into the developing
flowers and transfer the T-DNA. This then
results in the formation of transgenic seeds that
can be planted out on medium with a selectable
marker. The plants that survive on the
selectable medium get transplanted and can be
studies or screened.
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• Agrobacterium versus particle bombardment
• In general Agrobacterium is considered the method
  of choice for transformation.
• Advantages include
• low copy number of the transgene
• higher proportion of stable transformants
• larger DNA segments can be transferred
• more time-efficient
• Are there alternatives?
• YES!
• Electroporation of plant protoplasts/pollen/embryo
  s/callus. In this case the DNA is introduced
  while the cells are subjected to an electric
  discharge.
• Micro-injection. The DNA is injected into the
  protoplast with a pipet
• Silicon-carbide mediated transformation. DNA and
  silicon carbide fibers are mixed in with cells
  and mixed in a blender. The DNA-coated fibers
  penetrate the cells via small holes created by
  the fibers and introduce the DNA.
• Pollen-tube pathway. DNA can be applied to cut
  styles shortly after pollination and flows down
  the pollen tube to end up in the zygote.
• Liposomes. DNA is loaded into phospholipid
  spheres (liposomes) and these liposomes are mixed
  with protoplasts to result in lipofection

9
Did the transgene actually incorporate into the
genome? This is always the big question. The
selection based on a selectable marker is
supposed to result in plants that actually have
been transformed, but in some cases only the
marker gets transferred, but not the gene of
interest. It is possible to check for the
presence of the transgene by doing a Southern
analysis. Independent transformants are expected
to show hybridizing bands of different sizes. It
is also possible to check for integration with
anchored-PCR. The blot shows a Southern blot
with DNA from various independent transformants
of maize hybridized with a probed derived from
the transferred gene. Transformation of
maize We have seen comparisons between maize and
Arabidopsis a number of times (e.g. cloning
strategies). Agrobacterium has a fairly broad
host range but does not transfer DNA to monocots
(which include the agronomically important
grasses) very easily. The earliest attempts to
generate transgenic maize were therefore focuses
on the use of the particle gun. This meant
transformation of callus and required a lot of
time (4-6 months) at marginal success rates. It
is now possible to transform immature zygotic
embryos of maize with Agrobacterium. One of the
tricks is to add acetosyringone to the
co-cultivation medium (the medium in which the
bacteria and plant are mixed). This is a
compound secreted by various dicots that are
naturally susceptible to Agrobacterium and
stimulates the bacteria-plant interaction. The
addition of cysteine also helps. A patented
technique uses Agrobacterium with extra copies of
some of the vir genes. This improves
tranformation efficiency. The main disadvantage
is that very few lines can be used reliably for
transformation (inbred line A188 and hybrid Hi
II), but through regular crosses the transgene
can be introduced in other lines. The efficiency
is still not great, 5.5.
Frame et al. (2002)
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Maize transformation with Agrobacterium
From Frame et al (2002) Plant Phys 129 13-22