Lecture 18, Chapter 11 Analysis of transgenic plants

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Lecture 18, Chapter 11Analysis of transgenic
plants

• Neal Stewart

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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
  sulfatepolyacrylamide 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?

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Is my plant transgenic? How to know?

• Surviving selectionbut remember that there can
  be escapes. What other evidence?

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Using marker genes helps answers

• Are my plants transgenic?
• Is the gene expressed?
• How is my promoter working?

Negative selectable?
Positive selectable?
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Selectable markers Scorable markers (reporte
r genes)

• Typically used to recover transgenic plant cells
  from a sea of non-transgenic cells
• Antibiotic resistance markers and herbicide
  resistance markers are most common

• Can help visualize transient expression
• Can help visualize if tissue is stably transgenic
• Useful for cellular and ecological studies

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Sometimes escapes occur for kanamycin
resistance markers tissue is redvery stressed
Figure 9.3
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Is my plant transgenic?

• Surviving selectionbut remember that there can
  be escapes.
• Reporter genesbetter. But there must be a
  reporter gene in the vector. What works all the
  time?

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35SGFP canola
White light UV light in a darkened room
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Is my plant transgenic?

• Surviving selectionbut remember that there can
  be escapes. Not enough.
• Reporter genesbetter. But there must be a
  reporter gene in the vector.
• All around easy testPCR. But what if
  Agrobacterium survives in low amounts in the T0
  plants? Could give a false positive band. PCR is
  ok for biolistics.
• Can do PCR on T1 plants or look at segregation of
  the transgene.

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GFPBt segregation
Using GFP screening to see Bt when the
transgenes are linked. Nat Biotechnol 171125
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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 possiblebut most researchers
  are best convinced by Southern blot data.

Why all the mystique and skepticism?
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Good reasons for doubt

• New methods dont always work, but wishful
  thinking takes over (see Chapter 10 sectionthe
  Rush to Publish
• Resilient Agrobacterium can linger
• The unexpected can be tricky.
• Others?

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Southern blot analysis

• Gold standard for assessing transgenicity

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Southern blot steps

• Isolate genomic DNAmust be a lot and of good
  quality (not sheared)
• Restriction digestion (must be choosy about
  enzymeswell see why)
• Separate fragments on gel
• Transfer to nylon filter
• Probe filter with DNA of interest (transgene)

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Figure 6.1
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Restriction digest and gel electrophoresis
http//www.ndpteachers.org/perit/Electrophoresis2
05B25D.gif
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Southern blotDNA transfer to nylon
www.gbiosciences.com/Southern-Blot-desc.aspx
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Figure 11.4
Figure 11.4. Digestion of genomic DNA that is
electrophoretically separated for Southern blot
analysis (left) and a phosphorimage of the blot
after hybridization with radiolabeled probe
(right). Genomic DNA was extracted and digested
to completion and run on a 0.8 agarose gel
(left). The DNA was blotted to a membrane,
hybridized with a radiolabeled probe, and exposed
to a phosphor screen (right). There is a plasmid
band (.10 kb) in lane 1 (right) and one band in
lane 2 at 1.8 kb (right) that have sequences
complementary to the probe.
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Figure 11.6
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Figure 11.7
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Figure 11.4
What is missing in this experiment, or what would
you change?
Figure 11.4. Digestion of genomic DNA that is
electrophoretically separated for Southern blot
analysis (left) and a phosphorimage of the blot
after hybridization with radiolabeled probe
(right). Genomic DNA was extracted and digested
to completion and run on a 0.8 agarose gel
(left). The DNA was blotted to a membrane,
hybridized with a radiolabeled probe, and exposed
to a phosphor screen (right). There is a plasmid
band (.10 kb) in lane 1 (right) and one band in
lane 2 at 1.8 kb (right) that have sequences
complementary to the probe.
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Biotechnologist of the daySir Edwin M. Southern

• Southern blot 1975
• Invented it as a postdoc
• in Edinburgh (MRC)
• Needed to probe complex
• eukaryotic DNA (frog)
• for a specific sequence
• Worked the first time!

http//www.oxfordtoday.ox.ac.uk/2005-06/v18n2/01.s
html
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Southern blot analysis of transgenic plants with
an orange fluorescent protein

• Southern analysis of mOrange plants genomic DNA.
  A. Genomic DNA digested with BamHI,
  electrophoresed on 1 agarose Arabidopsis
  Lane 1. 21-12-3-3 Lane 2.
  21-12-6-1 Lane 3. 21-12-6-6
  Lane 4. 21-12-7
  Lane 5. 21-12-29
  Lane 6. 21-12-34 Lane 7.
  Columbia
• Tobacco Lane 1. 21-12-2-7
  Lane 2. 21-12-5-5 Lane
  3. 21-12-5-8 Lane 4.
  21-12-6-6 Lane 5.
  21-12-8-7 Lane 6. Xanthi
• plasmid pMDC32-mOrange (21-12) BamHI
  digest
• B. Transferred to nylon membrane.
  Hybridization to 32-P labeled probe of mOrange
  coding sequence. Exposed to phosphor screen 3
  weeks.

llg 2007-05-02 Sgel3 mOrange.tif
llg 2007-06-26 S mOrange 3wk.tif
Whats wrong?
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Southern on ST1 transgenics

• Southern analysis of Switchgrass genomic DNA.
  A. 10µg genomic DNA (CTAB 2x from leaf tissue)
  digested with XhoI, electrophoresed on 1.2
  agarose 2BR lines Panicum virgatum ST1 T0
  transgenic Pv ST1 nontransgenic
  control pANIC-2B/gbr15 plasmid XhoI digest
• Transferred to nylon membrane. Hybridization to
  32-P labeled probe of pporRFP coding sequence.
  Exposed to phosphor screen 6 day.

Whats wrong?
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Northern blot analysis

• Gives relative amount of gene expression-at the
  transcript level.
• Isolate mRNA be a lot and 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
Figure 11.9
What is missing in this experiment?