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. Therefore selection has been needed. – NPTII – Bar Recently, easily scorable and non-invasive markers.

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

Stable integration of transgene Transgene is permanently integrated into the genome of the host plant. Transmitted to progeny (T n plants) in Mendelian fashion Need convincing proof of stable integration Multiple assays are possible—but most researchers are best convinced by Southern blot data.

Fluorescent Proteins

PCR analysis by gel electrophoresis 500 bp 750 bp 1000 bp 1500 bp - + LadderSample

PCR and False Positives Genomic DNA Transgenic plant produced from Agrobacterium-mediated transformation In T 0 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.

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

Digested Genomic 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? LBRB DNA Probe EcoRI Site 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.

Why is a single cut within the T-DNA necessary? LBRB LBRB EcoRI Site 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.

Restriction digest and gel electrophoresis

Southern blot—DNA transfer to nylon

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)

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

Figure 11.9 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

Western blots and ELISAs often use amplification of signal via antibodies

Figure Western blot example What is missing in this experiment?

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.

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