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The two hardest things to handle in life
are failure and success.
Dr. Joyce Brothers

Pre-Lab Topics

• Reporter genes
• GUS staining

Experimental Procedures

• Staining of transgenic plants
• Poster critique

Overview of Procedures

Each team will choose an environmental condition (touch, cold, heat, hormone, drought) and compare staining in treated plants to the staining pattern for the same line that was not stimulated. Seedlings will be stained with X-GLUC in 12 or 24 well plates.

Stimulus Application: duplicate plates, one is treated and the other stays sealed on the bench during the treatment

• TOUCH: open plate and touch plants, wait 5 minutes, touch plants again, then wait 30 minutes
• COLD: surround sealed plate with ice for 10 minutes, then back on desktop for 30 minutes
• HEAT: put in 37°C incubator for 10 minutes, then back on desktop for 30 minutes
• HORMONE: open plate, spray with hormone, wait 40 minutes
• DROUGHT: open plate and wipe lid dry, move plants to lid for 10 minutes, then lay on top of media for 30 minutes
  
  Special Note: these are examples of conditions that are often studied; feel free to choose your own environmental stimulus to test (we'll let you know if it seems reasonable)

  ***What happens during the recovery time AFTER the stimulus?***

Reporter Genes

Reporter genes encode proteins whose expression is easily detected in a sensitive, specific, quantitative, reproducible, and rapid manner.
Some commonly used reporter genes include

• Chloramphenicol acetyltransferase (CAT)--a bacterial enzyme that transfers radioactive acetyl groups to chloramphenicol; detection involves thin layer chromatography and autoradiography (1).
• b-galactosidase (GAL)--a bacterial enzyme that hydrolyzes colorless galactosides to yield colored products.
• Luciferase (LUC)--a firefly enzyme that oxidizes luciferin and emits photons (2).
• Green fluorescent protein (GFP)--an autofluorescent jellyfish protein (3).
• b-glucuronidase (GUS)--an enzyme that hydrolyzes colorless glucuronides to yield colored products (4).

References
• Gorman C.M., Moffat L.F, and Howard b.H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2: 1044-1051.
• de Wet J.R., Wood K.V., DeLuca M. Helinsky D.R., and Subramani S. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7: 725-737.
• Tsien R.Y. (1998) The green fluorescent protein. Annu. Rev. Biochem. 67: 509-544.
• Jefferson R.A. (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387-405.
• Alam J. and Cook J.L. (1990) Reporter genes: Application to the study of mammalian gene transcription. Anal. Biochem. 188: 245-254.
• Bronstein I., Fortin J., Stanley P.E., Stewart G.S.A.B., and Kricka L.J. (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219: 169-181.
• Groskreutz D. and Schenborn E.T. (1997) Reporter systems. Methods Mol. Biol. 63: 11-30.

GUS staining

• X-GLUC Buffer
  • 125 ml 200 mM NaPO4, pH 7.0
  • 0.413 ml 10% K3[Fe(CN6)], or 1.25 ml of 0.1 M
  • 0.527 ml 10% K4[Fe(CN6)]x3H2O, or 1.25 ml 0.1 M
  • 5 ml 0.5 M EDTA
  • 0.25 ml 10% Triton X-100
  • 117 ml water
  • Filter sterilize
  • Store at 4°C, covered with foil (CN is light sensitive)
  
  
• 200 mM NaPO4, pH 7.0
  • Weigh out 8.66 g Na2HPO4 and 4.7 g NaH2PO4.
  • Bring total volume to 500 mL with Milli-Q water.
  • Check pH with paper.
  • Autoclave to sterilize.
  
  
• X-GLUC Staining Solution
  • Let the X-GLUC substrate sit at room temperature before mixing the staining solution.
  • Weigh out 1 mg X-GLUC per ml of staining solution.
    (Note: X-GLUC must be dissolved in 200 µl DMSO or DMF first--it will not dissolve in water.)
  • Dilute to proper volume with X-GLUC buffer.
  
  Staining Protocol
  1. Carefully remove the plant tissue and place into one well of a 12 or 24 well tissue culture plate containing 1-2 mL (enough to completely cover tissue) of X-GLUC staining solution.
  2. Vacuum infiltrate for 10 mins at 600 mmHg at room temperature.
  3. After releasing the vacuum slowly, allow tissue to incubate in the staining solution overnight to several days at 37°C; seal the plate with PARAFILM to prevent evaporation of the staining solution; cover the plate with foil or place in a dark chamber.

     YOU WILL HAVE TO COME IN THE DAY BEFORE LAB # 8 TO STOP THE STAINING REACTION AND CLEAR THE CHLOROPHYLL.

  4. Remove the staining solution. (CAREFUL! the buffer contains cyanide; dispose of ONLY in the labeled waste container.)
  5. Add 2 mL RO-water to stop the staining reaction.
  6. Let sit for 2-5 mins, then remove the water (dispose of in the sink, not the cyanide waste container).
  7. Immerse the tissue in 80-90% ethanol overnight at ROOM TEMPERATURE to bleach the tissue of chlorophyll; seal the plate with PARAFILM to prevent evaporation of the ethanol.
     *Alternatively, immerse the tissue in 100% ethanol for 15-30 min. and rinse several times with ethanol until the green tissue turns white.

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  Copyright, Acknowledgements, and Intended Use
  Created by B. Beason (bbeason@rice.edu), Rice University, 23 July 2003
  Updated 20 July 2011