40 Gravitational and Space Biology Volume 25 (1) Sept 2011 Short Communication Using Green Fluorescent Protein (GFP) Reporter Genes in RNAlater™ Fixed Tissue Anna-Lisa Paul 1 and Robert J. Ferl 1,2 1 Department of Horticultural Sciences and 2 Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32611 USA Plants in an orbital environment experience conditions that are distinctly unlike the earth-bound environments that have directed their evolution on Earth. This presents a unique opportunity to examine biological responses, particularly those involved in integrating gravity as a force shaping biological systems. One means of measuring these adaptive responses is to monitor the expression of genes that allow survival in those peculiar environments. In a recent series of spaceflight experiments conducted on the International Space Station (ISS) we utilized Arabidopsis thaliana (Arabidopsis) plants engineered with specific gene promoters driving to Green Fluorescent Protein (GFP). These biological sensor plants are referred to as TAGES, an acronym for Transgenic Arabidopsis Gene Expression System. One component of the TAGES Spaceflight experiment was the use of several different GFP reporter gene constructions to record tissue-specific changes in the patterns of gene expression in real time. Another component was the evaluation of genome-wide changes in these plants, a goal that required the use of the nucleic acid preservative RNAlater™ (Ambion). The utility of GFP gene reporters is well established (Haseloff & Amos, 1995; Manak et al., 2002; Paul et al., 2003; Paul et al., 2004; Sheen et al., 1995). The application to spaceflight is especially appealing as gene expression data can be collected telemetrically, thereby placing minimal demands on crew time. The use of RNAlater™ also offered the opportunity to evaluate the preservation of GFP for postflight analysis. In a series of preliminary experiments prior to launch, the TAGES plants were utilized in ground-based studies as part of a Payload Verification Test (PVT) to test the operations and science before the experiment was launched. One aspect that was evaluated was the responsiveness of the biosensor plants to environmental stress and the efficacy of using RNAlater™ to preserve evidence of GFP expression long after the stress response was incurred, in addition to its general use as an effective means to preserve the integrity of RNA. In the PVT, plates of TAGES plates were prepared with two or three genotypes and a variety of methods used to collect GFP expression data. Figure 1 shows GFP reporter gene plants grown vertically on nutrient agar plates. The TAGES ISS experiments utilized a variety of plant lines engineered to report on several aspects of the spaceflight environment, and among the plants shown in Figure 1, the left-hand side of each plate contains Adh::GFP plants, which respond to reduced levels of oxygen. The plants on the far right-hand side of the plate are used as positive controls (CaMV35s::GFP) and continuously express GFP throughout their cells. In the experiment shown A B C Figure 1. 100mm 2 Petri plate containing TAGES seedlings from the Payload Verification Test. The photograph on the left (A) was taken in white light, (B) shows the same plate photographed through a 510nm long pass filter in 488nm blue light with an Illumatool™ device. The native red fluorescence of chlorophyll is prominent in the Adh::GFP plants on the left side of the plate, and intense GFP expression can be seen in the positive control plants (CaMV35s::GFP) on the right side of the plate. The image in C was captured during PVT with the GFP Imaging System that is currently installed aboard the ISS.