Analysis of Transgenic Plants II

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Analysis of Transgenic Plants II
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Ultimately sequencing?
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Progeny Testing
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Segregation analysis of event 30b. Northern blot
analysisc. Root growth (trait)
Event number 27 28 29 30
All T1 generation
What can we infer about transgene expression of
events 28 and 30?
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Northern Analysis, Western Analysis
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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)

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Northern blot example
What is missing in this experiment?
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Western blot

• Also to measure gene expressionat 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

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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

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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 RT-PCR) 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

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http//www.rt-pcr.com/
Advantages of qRT-PCR over RT-PCR?
http//www.youtube.com/watch?vQVeVIM1yRMU
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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

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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
)
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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 reverse-orientation copy of the
  gene, an antisense copy designed to prevent or
  drastically reduce the formation of PG.

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Flavr Savr Tomato
Traditional Tomato
Flavr Savr is modified tomato for suiting modern
productions and distributions. Credit Owen Koo
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Napoli et al. The Plant Cell, Vol. 2, 279-289,
April 1990
Transgene 35S proCHSnos3
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.
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• 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.

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Result
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• 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.

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Northern blot
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• 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.

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• 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.

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• Early 1990s120,000 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.

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• May 1995Stephen 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.

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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.
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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.
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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.
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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.
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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.j
pg
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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

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Milestones in Food Biotechnology

• 1999 Gerber and Heinz baby foods GM-free
• 2000 Mc Donalds and Frito-Lay products GM-free

http//www.corrupt.org/articles/big_mac/bigmac.jpg
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Milestones in Food Biotechnology

• 2000 USDA Organic Foods Standards
• Must be GM-free

http//www.taquitos.net/im/sn/NaturalPlanet-Yellow
Corn.jpg
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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/_3
8232577_levy150.jpg
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The USDA has deregulated the first two Arctic
varieties, which can now be grown in the U.S.
without restriction.