Characterization of GFP gene expression using an automated image collection system and image analysis

Automated systems can be used to facilitate continual collection of biological information from a large number of samples over long periods of time. The proper combination of automated systems with fluorescent reporter genes such as the green fluorescent protein (GFP) offers the potential for analysis of gene expression over time without using destructive assays. Our goal was to develop an automated system to quantitatively evaluate and track both tissue growth and gene expression using the GFP reporter gene. The system consisted of a computer-controlled two-dimensional positioning table, a charged-coupled device camera mounted on a stereomicroscope equipped with a GFP fluorescence detection system. To keep the samples under aseptic conditions, the system was placed in a horizontal laminar-air flow hood.; Growth of two transgenic Arabidopsis clones was monitored for 10 days. Time-lapse animations showed that one clone had a wild type phenotype while the other clone exhibited a dwarf phenotype with delayed germination. Animations of the collected images showed regular “movements” of the plants, which clearly displayed circadian rhythm, nutation, and circumnutation events.; For gene expression analysis, the automated system was used to monitor expression of the mgfp5-ER and sgfp-TYG-35S genes in lima bean (Phaseolus lunatus L.) seeds for a 38 h period. Quantification of transient GFP expression showed that the sgfp-TYG gene was observed as early as 4 h after bombardment and reached a maximum expression at 24 h after bombardment. However, expression of the mgfp5-ER gene was first observed 7 h after bombardment and reached maximum expression after 24 h.; The image collection system was also used in long term experiments. GFP expression, driven by the lectin and 35S promoters in developing soybean somatic embryos, was monitored every 12 h for 28 days. Quantitative image analysis revealed that the lectin:gfp construction yielded low levels of expression during early stages of development but expression levels were similar to those from the 35S:gfp construction during late embryogenesis. The lectin:gfp construction showed a peak of expression 47 days after embryo development. The automated image collection system and image analysis were successfully used to track and quantitatively evaluate plant growth and gene expression.*; *This dissertation is multimedia (contains text and other applications not available in printed format). The accompanying CD requires the following system requirements: Quicktime.