Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: An assay amenable to high-throughput screening technologies

Abstract

Objective To identify genes that may participate in the pathophysiology of Sjögren's syndrome (SS), the technique of differential display was applied to labial minor salivary gland (MSG) biopsy samples. Methods Total RNA was isolated from MSG biopsy samples from a woman with primary SS and a control subject, and the differential display protocol with 8 different random oligonucleotide primers was performed. One particular differentially expressed fragment showed 98% homology with the cysteine‐rich secretory protein 3 (CRISP‐3) gene. The result was verified by reverse transcription‐polymerase chain reaction (RT‐PCR) with messenger RNA (mRNA) samples from MSG biopsy tissues obtained from 4 women with primary SS. A CRISP‐3 RNA probe was synthesized for in situ hybridization of 7 MSG biopsy samples from patients with primary SS. In an attempt to interpret the expression of CRISP‐3, normal peripheral blood lymphocytes (PBLs) were activated in vitro at different time points and assayed for CRISP‐3 expression. Finally, B cells were transfected with the coding region of CRISP‐3 and monitored for the up‐regulation of different B cell activation markers. Results The CRISP‐3 gene was detected by RT‐PCR in all SS patients tested. Mainly the mononuclear cells infiltrating the MSGs of patients expressed CRISP‐3 mRNA. In addition, CRISP‐3 was detected by RT‐PCR between 30 minutes and 6 hours in phorbol myristate acetate‐activated normal PBLs, while staurosporine inhibited this expression. CRISP‐3‐transfected B cells exhibited an up‐regulation in CD25 surface expression. Conclusion The CRISP‐3 gene is identified as a novel early response gene that may participate in the pathophysiology of the autoimmune lesions of SS.

Automated microscope system for determining factors that predict neuronal fate

“…Erb and colleagues (17) recently showed that the loss of GFP is a sensitive and specific marker of different types of death in some nonneuronal cell types. If GFP loss corresponds to the death of a neuron, it can be quantified over time as a measure of neuronal survival (17,18). To test this possibility, we simultaneously measured the loss of GFP in transfected neurons and the loss of membrane integrity by using a membrane-impermeant nuclear dye, EtHD.…”

“…Next, we tested whether the expression of GFP affected survival. Studies of the potential toxicity of GFP and its variants have arrived at conflicting conclusions (17)(18)(19)21). If GFP is toxic, the level of GFP expression as measured by GFP fluorescence should correlate inversely with duration of survival.…”

Detection of myeloma in skeleton of mice by whole-body optical fluorescence imaging

“…Optical imaging of GFP-expressing cancer cells offers a means of visualizing tumors in situ in whole intact animals (16,17), and we reasoned that combining the 5TGM1 myeloma model with GFP imaging would facilitate accurate monitoring of myeloma tumors disseminated throughout the skeleton. This capability is especially important in settings where the model is being used to predict efficacy of antitumor agents, as there is a marked decrease in emitted fluorescence observed following death induction in cells expressing eGFP (20).…”

“…Importantly, visualization of emitted fluorescence requires no preparative procedures, substrates, or contrast agents, and it is convenient and relatively inexpensive. Moreover, 5TGM1-eGFP myeloma cells could be used to develop high-throughput GFP-based microplate cytotoxicity screens for evaluation of novel anticancer agents, as shown for other eGFP-expressing tumor-derived cells (20). In vivo efficacy of positive ''hits'' could then be rapidly determined in mice inoculated with the same 5TGM1-eGFP cells as we have shown in this report.…”

High-Throughput Growth Assay forToxoplasma gondiiUsing Yellow Fluorescent Protein

“…However, all of these assays employ FACS to assess drug activity and as such are limited in the number of compounds that can be tested during a given time period. Combining GFP with a microtiter format enhances the throughput of the screening system considerably and has recently been applied to determine the activities of anticancer drugs (37,47), but to our knowledge it has not as yet been applied for antimicrobial drug screens.…”