Lab 11
Reporter Genes
GUS
The GUS gene is used in transgenic studies to verify the transmission of the transformation vector into the target
plant. It is informative because nothing in the plant tissue would stain blue; the blue stain can only mean the
plant is transformed. GUS makes a protein that turns blue when mixed with X-gluc (5-Brom-4-chlor-3-indolylß-Dglucuronide). Chimeras can be observed in the X-gluc stained tissue.
X-gluc stain (prepared):
2.5ml
0.2M Na2HPO4
2.4ml
100µl
D.I. water
0.5M Na2EDTA
5.0mg X-gluc
5ml
1. Put 30-40µL fresh X-gluc stain into the well of a multi-well tissue culture plate. You will use one plate per
two people. Count the number of shoots you have from your transgenic tobacco- this is the number of wells
you’ll need to add GUS stain to.
2. Cut a thin section of leaf or stem and place it into an individual well - keep track of where sections come from
in your notebook.
3. Stain sections for 4-5 hr. at 37 in the incubator.
4. After staining, clear or fix the tissue by adding 50µL of 95% ethanol:glacial acetic acid (3:1 v/v). Wait a half
hour before scoring for GUS. Perform step under Hood. You will have to come back on Friday to see your
results. The fixing solution will be added for you in the morning.
5. Under the dissecting microscope, score the tissues for GUS (blue) staining. Note full GUS and chimera
tissues. Glacial acetic acid can damage microscopes - leave the cover on the microtiter plate.
GFP
GFP, or green fluorescence protein, is becoming the reporter gene of choice for transgenic studies. As the name
implies, the GFP gene product will fluoresce green under UV light. Using GFP allows plants to be scored
without damaging the tissue. Entire plant organs can be viewed under the dissecting scope equipped with
fluorescence capability.
For this lab section, we will look at GFP positive material provided from another lab, TBD.
NPTII
MS media with Kanamycin has been prepared for you. You will be given a small quantity of Arabidopsis seeds
from your ‘Dip’ experiment. Only transformed seeds should sprout on the kanamycin selection.