Analysis of Transgenic Plants

1.Regeneration on Selective Medium Selectable Marker Gene

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

Sometimes “escapes” occur

Scorable Marker Gene

GUS

GFP Cloned from the jellyfish Aequoria victoria

GFP 27 kDa monomer that fluorescences green under UV (365nm) or blue (490) light

GFP Must be highly expressed

Luciferase

Bar gene

Fluorescent Proteins

Phenotype

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

PCR and False Positives Genomic DNA Transgenic plant produced from Agrobacterium-mediated transformation In T 0 plants, Agrobacterium left over from the initial transformation may still be present in the plant tissue. 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 DNA probe

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)

Southern Analysis

Digested Genomic 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.

Southern blot—DNA transfer to nylon

Ultimately sequencing?

Progeny Testing

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 All T 1 generation

Northern Analysis, Western Analysis

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)

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

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

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

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. Napoli et al. The Plant Cell, Vol. 2, , April 1990 Transgene: 35S pro::CHS::nos3’