1. Lecture 19, Chapter 11 Analysis of transgenic plants part II Neal Stewart

2. 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?

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

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

5. 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.

6. Figure 11.6

7. Figure 11.7 A Southern blot could have two objectives: 1.Is the gene of interest intact? Solution: used HindIII and look for 800 bp fragment (see previous slide) 2.How many insertions (copies are there)? Solution: Use EcoRI or SacI and count the number of fragments (of assorted sizes)

8. 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

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

10. 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

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

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

13. For all blots (and all assays for that matter) Use appropriate controls, such as a non- transgenic 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

14. Figure 11.2 ELISA—Enzyme-linked immunosorbant assay

15. Real-life example 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

16. How ABC WBC19 might work

17. Are the plants transgenic for the ABC transporter? Digested with SacI and KpnI and probed with the ABC transporter

18. a.Segregation analysis of event 30 b. Northern blot analysis c. Root growth (trait) What can we infer about transgene expression of events 28 and 30? Event number 27 28 29 30 All T 1 generation

19. PCR-based methods of gene expression analysis—transcript (mRNA) level Is my transgene expressed? Reverse transcriptase PCR (RT-PCR)— recall when reverse transcriptase was used in an earlier lecture? Real-time RT-PCR also known as quantitative RT-PCR (qRT-PCR) Why use PCR techniques instead of blotting experiments (e.g., Northern blot analysis) to estimate transgene expression?

20. 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 RT- PCR kit is better. What is the basis of their case?

21. 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.

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

23. 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?