Analysis of Transgenic Plants

1. Regeneration on Selective MediumSelectable 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

http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg

Phenotype

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

PCR and False Positives

Genomic DNATransgenic plant produced from Agrobacterium-mediated transformation

• In T0 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?

LB RB

DNA ProbeEcoRI 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

www.gbiosciences.com/Southern-Blot-desc.aspx

Ultimately sequencing?

Progeny Testing

a. Segregation analysis of event 30b. Northern blot analysis

c. Root growth (trait)

What can we infer about transgene expression of events 28 and 30?

Event number27 28 29 30

All T1 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

http://www.youtube.com/watch?v=QVeVIM1yRMU

http://www.rt-pcr.com/ 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, 279-289, April 1990

Transgene: 35S pro::CHS::nos3’