Analysis of Transgenic Plants II
Ultimately sequencing?
Progeny Testing
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?
Northern Analysis,
Western Analysis
Northern blot analysis
• Gives relative amount of gene expression-at
the transcript level
• Isolate mRNA of good quality (not degraded)
• Separate transcripts on a gel
• Transfer to nylon filter
• Probe filter with DNA of interest (transgene)
Northern blot example
What is missing in this experiment?
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
RT-PCR
•
•
•
•
Isolate RNA from tissues of interest
Eliminate all DNA from a sample
Make cDNA from mRNA
Perform PCR on sample using
transgene-specific primers
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?
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
Why GM Food?
[2]
- Example
• The first GM whole food, FLAVR SAVRTM tomato, was
marketed in the United States in 1994.
– Slower ripen rate
– Ripen longer on vine
– Fully developed flavors
Flavr Savr Tomato developed by Calgene
(Sources: http://www.ca.uky.edu/agripedia/glossary/flavr.htm)
Calgene
• Circumstantial evidence available in the 1980s
suggested that expression of the tomato fruit
enzyme polygalacturonase (PG) was key to
fruit softening.
• Researchers at Calgene, Inc., in Davis,
proposed to suppress PG accumulation in
ripening tomatoes by introducing a reverseorientation copy of the gene, an “antisense”
copy designed to prevent or drastically reduce
the formation of PG.
Flavr Savr Tomato
Traditional Tomato
The Flavr Savr
tomato ripens
on the vine –
resulting in
fuller flavour.
It is modified so
that it remains
firm after
harvesting
The traditional
tomato must be
harvested while
it is still green
and firm so that
it is not crushed
on the way to the
supermarket.
The traditional
tomato is sprayed
with ethylene
after shipping to
induce ripening.
Ripe
and
Increas
ed
Flavour
.
Supermarke
t
Ripe
but
decrea
sed
flavou
r.
Flavr Savr is modified tomato for suiting modern productions and distributions. Credit: Owen Koo
Napoli et al. The Plant Cell, Vol. 2, 279-289, April 1990
Transgene: 35S pro::CHS::nos3’
Transgenic line 1
Transgenic line 2
Transgenic line 3
Transgenic line 4
Phenotypes of chimeric CHS transgenotes and variations among flowers on single
plants. A control (parental) flower is shown along with four different CHS
transgenotes.
• By 1987, Calgene researchers identified and
cloned a tomato fruit PG gene, developed
methods for tomato transformation and
regeneration, and produced tomato plants
with inserted PG antisense DNA constructions.
• In October 1992 the U.S. Department of
Agriculture determined that the PG-antisense
tomato lines were not a “plant-pest” risk and
no longer required permits for field testing or
transport.
Result
• On May 21, 1994, the genetically engineered
FLAVR SAVR tomato was introduced in Davis
and Chicago.
• Demand for this product was high and
remained high, but the product was never
profitable because of high production and
distribution costs.
Northern blot
• In 1996, Zeneca, under license, introduced in
the United Kingdom paste from PG-antisense
tomatoes grown and processed in California,
in collaboration with the grocery chains
Sainsbury's and Safeway.
• More than 1.8 million cans, clearly labeled as
derived from genetically engineered
tomatoes, were sold from 1996 through early
1999.
• 1986
Cattle in Britain begin to suffer from a
condition similar to scrapie in sheep,
nicknamed “mad cow disease” due to the
behavior of the sick cows. The cause is
unknown, though some suspect the feeding of
rendered scrapie-infected sheep to cattle.
• Early 1990s
120,000 cattle have been diagnosed with BSE
in Britain. The British government insists the
disease poses no threat to humans. House
cats begin dying from beef byproducts in their
pet food. Five types of antelope die in British
zoos from TSEs that had been fed commercial
cattle feed. Through all of this, the British
government continues to adamantly insist
that British beef is perfectly safe, and BSE is no
threat to humans.
• May 1995
Stephen Churchill, 19, becomes the first victim
of a new version of Creutzfeldt-Jakob Disease
(vCJD). His is one of three vCJD deaths in
1995.
• July 1997
21 vCJD victims in Britain have been
confirmed, many more unconfirmed cases.
SOURCE: Fernandez-Cornejo, J., Wechsler, S., Livingston, M. and Mitchell, L. 2014.
Genetically Engineered Crops in the United States. USDA Economic Research Service Report
No. 162, February 2014.
SOURCE: Fernandez-Cornejo, J., Wechsler, S., Livingston, M. and Mitchell, L. 2014.
Genetically Engineered Crops in the United States. USDA Economic Research Service Report
No. 162, February 2014.
SOURCE: Fernandez-Cornejo, J., Wechsler, S., Livingston, M. and Mitchell, L. 2014.
Genetically Engineered Crops in the United States. USDA Economic Research Service Report
No. 162, February 2014.
SOURCE: Fernandez-Cornejo, J., Wechsler, S., Livingston, M. and Mitchell, L. 2014.
Genetically Engineered Crops in the United States. USDA Economic Research Service Report
No. 162, February 2014.
Milestones in Food
Biotechnology
• 1999: GM corn and
soybean products are
present in 80% of
processed foods in USA
– Corn:
• starch, high fructose
corn syrup, oil
– Soy:
• oil, Lecithin, protein
http://nadav.harel.org.il/cola/image/CokeClassic.jpg
Milestones in Food Biotechnology
• 1999: European Union requires
GM labels
– blocks import of GM corn, beans
• Ban lifted 2004
– but no change in anti-GM
sentiment in Europe
– Affects African export crops
• Paternalism
Milestones in Food
Biotechnology
• 1999: Gerber and
Heinz baby foods GMfree
• 2000: Mc Donalds and
Frito-Lay products
GM-free
http://www.corrupt.org/articles/big_mac/bigmac.jpg
Milestones in Food
Biotechnology
• 2000: USDA Organic
Foods Standards
– Must be GM-free
http://www.taquitos.net/im/sn/NaturalPlanet-YellowCorn.jpg
Milestones in Food Biotechnology
• 2002 Zambia refuses
GM maize as food aid
– To help 2.5 million in
food shortage
– Calls GM food “poison”
– Heavily influenced by
European attitudes
about GM
Zambian President Mwanawasa
http://news.bbc.co.uk/media/images/38232000/jpg/_38232577_levy150.jpg
The USDA has deregulated the first two Arctic varieties,
which can now be grown in the U.S. without restriction.