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Lecture 18, Chapter 11 Analysis of transgenic plants part I

Lecture 18, Chapter 11 Analysis of transgenic plants part I. Mat Halter 3/27/12 Plant Genetics, Breeding and Biotechnology (PLSC 452/552), University of Tennessee. Lecture 19, Chapter 11 Analysis of transgenic plants part II. Neal Stewart. Transformation is a relatively rare event .

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Lecture 18, Chapter 11 Analysis of transgenic plants part I

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  1. Lecture 18, Chapter 11Analysis of transgenic plantspart I Mat Halter 3/27/12 Plant Genetics, Breeding and Biotechnology (PLSC 452/552), University of Tennessee

  2. Lecture 19, Chapter 11Analysis of transgenic plantspart II Neal Stewart

  3. Transformation is a relatively rare event. • Therefore selection has been needed. • NPTII • Bar • Recently, easily scorable and non-invasive markers.

  4. Figure 9.3 Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed

  5. 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.

  6. Fluorescent Proteins http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg

  7. PCR analysis by gel electrophoresis - Ladder Sample 1500 bp 1000 bp 750 bp 500 bp +

  8. PCR and False Positives Genomic DNA Transgenic plant produced from Agrobacterium-mediated transformation • 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.

  9. 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

  10. 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? Digested Genomic 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 T-DNA.

  11. Why is a single cut within the T-DNA necessary? EcoRI Site EcoRI Site LB RB LB RB RB If there is no EcoRI site within the tDNA, 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.

  12. Restriction digest and gel electrophoresis http://www.ndpteachers.org/perit/Electrophoresis%20%5B2%5D.gif

  13. Southern blot—DNA transfer to nylon www.gbiosciences.com/Southern-Blot-desc.aspx

  14. 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) http://www.youtube.com/watch?v=KfHZFyADnNg

  15. Northern Blot No digestion necessary… why is this? RNA loading controls are necessary to ensure an equal amount of RNA is loaded in each well.

  16. Northern blot example Figure 11.9 What is missing in this experiment?

  17. 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

  18. Western blots and ELISAs often use amplification of signal via antibodies http://probes.invitrogen.com/handbook/images/g001474.gif

  19. Western blot example Figure 11.11 What is missing in this experiment?

  20. 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.

  21. http://www.rt-pcr.com/ Advantages of qRT-PCR over RT-PCR? http://www.youtube.com/watch?v=QVeVIM1yRMU

  22. 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 Summary

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