GFP-Based Methods for Detecting Apoptosis
Apoptosis, also called programmed cell death, is a very important cellular process that plays a vital role in maintaining the normal physiological function of the organism. It must be precisely regulated in order to maintain the proper functioning of our body. At present, there exist a variety of techniques that can detect the process at different stages. However, all of these techniques have certain limitations. Therefore there is a strong need to develop efficient methods that can detect the activation of the apoptotic process in the living cells. Unlike conventional techniques, the invention provides a highly sensitive yet simple assay that detects the early stages of apoptosis. The detection method of this invention is based on the fact that the process of apoptosis involves the activation of a series of intracellular proteases called "caspases". It provides a fluorescent protein construct to detect protease or caspase activated apoptosis. The construct contains a donor fluorescent protein, an acceptor fluorescent protein and a linker peptide positioned in between the donor and acceptor. The linker peptide contains a substrate sequence of a caspase. This invention provides nucleic acids encoding the fluorescent protein construct, expression constructs comprising the nucleic acids as well as gene delivery vehicles containing these nucleic acids. It also provides tools to facilitate future studies leading to a better understanding of the mechanisms regulating apoptosis and will assist in the discovery and development of new drugs to combat many important diseases. Those diseases related defective or excessive apoptosis may included cancer, AIDS, auto-immune diseases and neurodegenarative diseases.
1. This probe can be used in both in vivo and in vitro assay for apoptosis
2. The detection procedure is simple
3. Useful in large scale screening of drugs
4. No special procedure is required
- Laboratories in the world studying apoptosis and related diseases
- Research conducted in scientific institutes or in the pharmaceutical industry for high throughput anti-cancer drug screening
Patent Number: CN1254546C
Application Number: CN2002120427A
Inventor: ZHANG D | LUO Q
Priority Date: 24 May 2001
Priority Number: CN1254546C
Application Date: 24 May 2002
Publication Date: 3 May 2006
IPC Current: C12Q000104 | C12Q000125 | C12Q000137
Assignee Applicant: The Hong Kong University of Science & Technology
Title: Process based on green fluoresin for detecting cell wither | Method based on green fluorescent protein detecting cell apoptosis
Usefulness: Process based on green fluoresin for detecting cell wither | Method based on green fluorescent protein detecting cell apoptosis
Summary: (IV) containing (I) or (II) is useful for detecting caspase or protease activated apoptosis which involves culturing (IV) containing (I) or (II) under conditions suitable for excitation of the donor fluorescent protein and detecting a fluorescent property in the sample, where the presence of caspase activated apoptosis or programmed cell death in the cells of the sample results in a change in the degree of fluorescence resonance energy transfer between the donor protein and the acceptor protein. The method further involves quantification of the change in the degree of fluorescent energy transfer. (IV) containing (I) or (II) is useful for determining whether an agent modifies caspase or protease activated apoptosis in a host which involves contacting (IV) containing (I) or (II) with the agent and culturing the cells under conditions suitable for excitation of the donor fluorescent protein and determining a fluorescent property of the sample, where an activity of the agent is determined by a change in the degree of the fluorescent property in the presence and absence of the agent. The agent is chosen from small molecule compound, protein, nucleic acid, ribozyme, antisense nucleic acid and isolated compounds from traditional Chinese medicine and their chemical derivatives. The method further involves providing a second sample of host cells and culturing the second sample under conditions suitable for exciting the donor fluorescent protein and comparing the degree of fluorescence resonance energy transfer in two samples (all claimed).
Novelty: Fluorescent protein construct useful for detecting caspase or protease activated apoptosis, having donor and acceptor fluorescent protein linked by peptide linker with substrate sequence of caspase or protease
生物医学
生物工程
24 May 2002
Chinese 02120427.6
Chinese ZL02120427.6
TTC.PA.153
香港
