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 use of an automated system and image analysis would allow semi-continual monitoring and non-invasive quantification of the green fluorescent protein (GFP) expression and would therefore provide a better assessment of the levels of gfp gene expression than monitoring GFP at large time intervals. The main aim of this research was to monitor and quantify the expression of the gfp gene from the jellyfish (Aequorea victoria) and in vitro plant growth over time using an automated image acquisition system in combination with image analysis. The system, developed over the course of this work, consisted of a computer controlled two-dimensional positioning table and a charged-coupled device (CCD) camera mounted on a stereomicroscope equipped with a GFP fluorescence detection system. The image collection system was placed in a horizontal laminar air flow hood to provide an aseptic environment for monitoring in vitro cultures. In order to compare the pattern of expression of a soluble and an endoplasmic reticulum-targeted gfp gene, images of lima bean (Phaseolus lunatus L.) cotyledons transiently expressing the two different gfp genes, were collected every 30 min for 38 h. Time-lapse animations together with quantification of transient GFP expression using image analysis, showed that expression of the cytoplasmic soluble gfp gene was detected as early as 4 h after bombardment and reached a maximum at 24 h after bombardment. Expression of the endoplasmatic reticulum-targeted gfp gene was first observed 8 h after bombardment and reached its maximum expression after 24 h. The pattern of GFP expression, driven by the soybean lectin and 35S promoters, was monitored every 12 h for 28 d during somatic embryo development using the automated image collection system. Gene expression was then quantified using image analysis. Quantitative analysis revealed that, even though the lectin: gfp construction showed low levels of expression during early stages of development, expression levels eventually reached levels similar to those recorded from the 35S: gfp construction. Embryos with gfp under the regulatory control of the lectin promoter showed a peak of expression 47 days after embryo development, while GFP expression driven by the 35S promoter gradually increased throughout embryo development. Time-lapse animations were useful in characterization of gfp expression, and revealed a high variability in levels of gfp expression driven by the 35S promoter. Southern analysis showed the presence of multiple copies of the introduced plasmids for clones generated using particle bombardment. The copy number of clones containing the lectin: gfp construction, was not correlated with levels of gfp expression; however, a high copy number may have led to reduced levels of GFP expression of a clone containing the 35S: gfp construction.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:plant transformation analysis of gene expression image robotics green fluorescent protein lima beans soybean wheat arabidopsis
Date of Publication:01/01/2002