Analysis of Transgenic Plants. 1.Regeneration on Selective Medium Selectable Marker Gene.
Transcript of 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.
GUS
Fluorescent Proteins
http://en.wikipedia.org/wiki/File:FPbeachTsien.jpg
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)
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
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 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)
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’