Lecture 12
Analysis of transgenic plants
Neal Stewart and Mat Halter
Discussion questions
1. What are the established methods to determine if a plant is transgenic and
whether the transgene(s) is expressed?
2. In a Southern or northern blot, through what type of chemical bond does
the complementary probe bind to nucleic acid?
3. Nucleic acids and proteins are separated according to size in agarose and
sodium dodecyl sulfate–polyacrylamide gel electrophoreisis (SDS-PAGE)
gels, respectively. Why do both types of macromolecules migrate
toward the anode in an electrical current?
4. What is gene expression, and how can you measure it?
5. Explain why phenotypic data provide evidence of transformation but not
proof of a transformation event.
6. What factors are most important when designing a Southern blot
experiment to test for transgenic status?
Is my plant transgenic?
Fig 12.1
LB
P
Visual
Selection
Visual Selection
(OFP)
T
P
Antibiotic
Selection
T
P
Gene of
Interest
T
Antibiotic Selection
(Hygromycin)
Molecular methods
for transgene
insertion, copy
number, and
expression as well
as Mendelian
segregation of
transgene in
progeny
RB
Fluorescent Proteins
http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg
Visual selection using FPs
T+
T-
T-
T+
Antibiotic Selection
• When a mixture of transformed and
untransformed callus is placed on antibiotic
selection media, only the transformed callus
carrying the antibiotic selection cassette is
able to survive and grow.
• In most cases, the untransformed callus dies,
making it “easy” to select for callus carrying
the T-DNA.
Sometimes “escapes” occur– for kanamycin
resistance markers tissue is red—very stressed
Is my plant transgenic?
• Survives selection with antibiotics or herbicide—but
remember that there can be escapes. Need more
proof besides surviving selection.
• Reporter genes—better. But there must be a reporter
gene in the vector. What about false positives?
• All around easy test—PCR. But what if
Agrobacterium survives in low amounts in the T0
plants? Could give a false positive band. PCR is ok for
biolistics. Why?
• Can do PCR on T1 plants or look at segregation of the
transgene.
GFP+Bt segregation
Using GFP screening to “see” Bt when the transgenes are linked. Nat Biotechnol 17:1125
Stable integration of transgene
• Transgene is permanently integrated into the
genome of the host plant.
• Transmitted to progeny (Tn plants) in
Mendelian fashion
• Need convincing proof of stable integration
• Multiple assays are possible—but most
researchers are best convinced by Southern
blot data.
Why all the mystique and skepticism?
Good reasons for doubt
• New methods don’t always work, but wishful
thinking takes over (see Chapter 11 section—
the Rush to Publish)
• Resilient Agrobacterium can linger
• Others?
Molecular characterization of
transgenic plants
• PCR- Simplest and fastest method. Prone to false
positives.
• Southern Blot- Confirms insertion of the T-DNA into the
genomic DNA of the target organism, as well as
provides insertion copy number.
• Northern Blot- Confirms the presence of RNA transcript
accumulation from the transgene of interest.
• Western Blot- Confirms presence of the PROTEIN
produced from the inserted transgene of interest.
• qRT-PCR- Provides a relative expression level for the
gene of interest—transcript—like Northern blot.
PCR and
DNA Gel Electrophoresis
PCR- Polymerase chain reaction, uses DNA primers to
amplify a target sequence of DNA, producing billions of
copies of identical DNA.
Gene cloning
Molecular
analysis
(Confirmation of the presence
of a particular fragment of
DNA in a pool of DNA)
-
PCR analysis by gel electrophoresis
= do PCR and run DNA on a gel
-
-
+
+
+
+
Gel electrophoresis
• The migration of DNA through an agarose matrix using the
application of an electric field.
• Agarose, when solidified in a gelatin form, produces a thick
netting that allows small particles to move through it quickly,
while larger particles move more slowly.
• By moving particles of different size through the agarose gel,
they can be separated, with the small particles moving quickly
away from the slower moving large particles.
• This method is used to separate DNA fragments by size.
PCR analysis by gel electrophoresis
-
Ladder
1500 bp
1000 bp
750 bp
500 bp
+
Sample
PCR and False Positives
Transgenic plant produced from
Agrobacterium-mediated
transformation
Genomic DNA
• In T0 plants, Agrobacterium left over from the initial
transformation is still present in all tissues.
• Contamination of the genomic DNA with the initial transformation
vector that is still present in the agrobacterium can produce a PCR
band.
Southern Blot
• Southern blotting confirms the presence of
the gene of interest in the genomic DNA of the
target plant and avoids the pitfalls of potential
false positives.
• Steps
– Genomic DNA isolation
– Restriction enzyme digestion of genomic DNA
– Running digested DNA on agarose gel to separate
fragmented DNA by size.
– Transfer of separated DNA to nylon membrane
– Hybridization with radioactive DNA probe
Restriction digest and gel electrophoresis
http://www.ndpteachers.org/perit/Electrophoresis%20%5B2%5D.gif
Restriction Digestion of Genomic DNA
• Restriction digestion of genomic DNA produces a
streak on an agarose gel rather than a single band.
Why?
Example: EcoRI
• What is the probability of a sequence of DNA in a plant
genome having the sequence of bases corresponding to an
EcoRI cut site?
• Each site can be 4 possible bases (A, T, C, or G), and the EcoRI
enzyme requires 6 sites (GAATTC)
• The probability of finding a random site in a genome that
happens to have the sequence GAATTC can be calculated:
1⁄4 x 1⁄4 x 1⁄4 x 1⁄4 x 1⁄4 x 1⁄4 = 1⁄4096
• Probability states that there will be an EcoRI cut site once
every 4096 bases, purely by chance.
EcoRI example, cont.
• The Arabidopsis thaliana genome is roughly 157,000,000 base
pairs in size.
157,000,000⁄4096 = 38,330
• Though this value is only based on probability, and therefore
may not be the TRUE number of EcoRI cut sites in this
genome, it can still accurately be assumed that there are A
LOT of cut sites.
• If restriction digested with EcoRI, the Arabidopsis genome
would be cut into tens of thousands of pieces, all of unique
size.
• This is why when you run a sample of digested genomic DNA,
you see a streak, rather than a band. The streak shows all
sizes of DNA produced by the random assortment of cut sites
within the genome.
Digested Genomic
• Essentially, every known
restriction enzyme will have cut
sites in a plant genome.
• How can enzyme selection be
used to detect copies of an
inserted transgene?
EcoRI Site
DNA Probe
LB
RB
• Single cutting enzymes can be
designed into the T-DNA before
transformation that will enable
proper digestion of the genome as
well as a single cut within the TDNA.
Figure 12.4
Southern Blotting
Isolated genomic DNA from transgenic plant
Gel electrophoresis
Restriction enzyme digest
-
+
Southern Blotting
Transfer separated DNA from agarose gel to nylon membrane
Agarose Gel
Nylon Membrane
Hybridizing the DNA probe
• The DNA probe is designed to be
complimentary to your gene of
interest.
•It is synthesized using radioactive
phosphorus, which emits a
detectable signal.
•The complimentary probe will
bind (by hydrogen bonding) only
to your gene of interest because of
the high sequence specificity.
Southern Blotting
Southern Blotting
Lane 1- Ladder
Lane 2- Negative Control
Lanes 3-8- Experimental Events
• Bands at different places
from event to event indicate
insertion at different places
in the genome.
• The number of bands in
each well indicates how
many insertions there were
in each event.
Final blot
Why is a single cut within the T-DNA
necessary?
EcoRI Site
EcoRI Site
LB
RB
LB
RB
If there is no EcoRI site within the T-DNA, after digestion
with EcoRI these two insertion sites will be indistinguishable
from one another after electrophoresis and probing.
Cutting within the T-DNA is necessary to distinguish each
and every insertion event. This is VERY important.
Restriction enzyme selection for Southern
blotting
BamHI EcoRI
LB
P
Visual
Selection
EcoRI
T
BamHI
P
Antibiotic
Selection
T
P
EcoRI
Gene of
Interest
Probe
BamHI
T
RB
Size (kb)
10
6
4
3
2
1.5
M WT 1
2
3
4
5
6
P
Northern Blot
• Confirms the presence of mRNA transcripts
transcribed from the gene of interest in the target
organism.
• Extremely similar to the Southern blot, but
detects RNA instead of DNA.
• Steps:
–
–
–
–
Isolation of RNA
Running RNA on agarose gel to separate by size
Transfer separated RNA from gel to membrane
Hybridize a radioactive DNA probe to the RNA on the
membrane
Sound familiar?
Northern Blot
No digestion with
RE is necessary…
why is this?
RNA loading controls
are necessary to
ensure an equal
amount of RNA is
loaded in each well.
Western blot
• Also to measure gene expression—at the
protein level.
• Extract proteins
• Separate proteins on a vertical gel
• Transfer to a membrane using an
electrotransfer system
• Probe with antibodies.
• Stain for antibodies
Western blots and ELISAs often use
amplification of signal via antibodies
http://probes.invitrogen.com/handbook/images/g001474.gif
Figure 12.11
Western blot example
What is missing in this experiment?
For all blots (and all assays for that
matter)
• Use appropriate controls, such as a nontransgenic plant (negative) and a positive
control typically plasmid for Southerns and
specific for westerns.
• Use an appropriate standard or a range of
standards.
• Set up the experiment intelligently
ELISA—Enzyme-linked immunosorbant
assay
Real-life example
• Can we engineer plants with a plant gene as a
kanamycin resistance selectable marker?
• An ABC (ATP-binding cassette) transporter
from Arabidopsis
• Used to produce transgenic tobacco
• Compared against nptII gene
• Both regulated by 35S promoters
Mentewab and Stewart, 2005, Nature Biotechnology 23:1177-1180
How ABC WBC19 might work
Are the plants transgenic for the ABC
transporter?
Digested with SacI and KpnI and probed with the ABC transporter DNA
a.
Segregation analysis of event 30
b. Northern blot analysis
c. Root growth (trait)
Event number
27 28 29 30
All T1 generation
What can we infer about transgene expression of events 28 and 30?
RT-PCR
• Isolate RNA from tissues of
interest
• Eliminate all DNA from a
sample
• Make cDNA from mRNA—
what is the result?
• Perform PCR on sample
using transgene-specific
primers
The figure is from an advertisement from a
company—they are making the case that their RTPCR kit is better. What is the basis of their case?
Real-time PCR or Quantitative PCR
• Real-time PCR uses fluorescence as an output
for DNA amplification in real-time.
• The amount of starting template DNA (or
cDNA for RNA measurement (real-time RTPCR) is correlated with the Ct number.
• More DNA = lower Ct; Ct is the cycle number
when a threshold amount of DNA is produced
during the PCR experiment.
http://www.rt-pcr.com/
http://www.youtube.com/watch?v=QVeVIM1yRMU
Advantages of qRT-PCR
over RT-PCR?
Fig. 12.3
A
B
C
Summary
• Is my plant transgenic?
– Survives selection
– Reporter gene
expression
– Progeny analysis
– PCR
– Southern blot analysis
• Is my plant expressing
the transgene?
–
–
–
–
–
Northern blot analysis
Western blot analysis
ELISA
RT-PCR
Real-time RT PCR
If you could choose just 3 of the above
analyses, which ones would you choose
and why?