A Novel System for Measuring Protease Activity
- 技術優勢
- The system is highly flexible. It can:be designed to detect virtually any protease activity of interest;be designed to target proteases located in membranes or vesicles; andaccommodate any measurable reporter protein. The system can be used to observe proteolytic activity in vitro and in vivo in both healthy cells and diseased cells. The system appears to work in a wide range of cell-types. The system can be used to generate transgenic animals to permit study of proteases of interest in whole organisms. The system works in transgenic organisms such as zebrafish, a vertebrate model that allows genetic screening and in vivo studies. With its rapid development and transparent embryo, zebrafish are amenable to large-scale genetic screens in 96-well plates. Thus, high throughput drug screening for proteases modulators is possible in an intact vertebrate animal model using this invention.
- 技術應用
- The invention may be used:As an efficient bioassay to determine the role of proteases in both normal and diseased cellular processes in biologically relevant environmentsTo screen for pharmacologic modulators of proteases
- 詳細技術說明
- The present invention provides a novel way to detect virtually any protease activity in cell cultures and in whole organisms. It can be used to screen for drugs or genes modulating a specific proteolytic activity. The system is based on DNA constructs, such as plasmids. It uses modular components, which makes it highly flexible. The system can be designed to detect any protease of interest. The DNA expression system can be delivered to cultured cells or used to generate transgenic organisms such as mice or zebrafish. Protease activity is directly related to a built-in reporter molecule.
- *Abstract
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None
- *IP Issue Date
- Aug 12, 2008
- *Principal Investigation
-
Name: Christopher Evans
Department:
Name: Amaud Lacoste
Department:
- 附加資料
- Patent Number: US7410765B2
Application Number: US2005281644A
Inventor: Lacoste, Arnaud | Evans, Christopher J.
Priority Date: 17 Nov 2004
Priority Number: US7410765B2
Application Date: 17 Nov 2005
Publication Date: 12 Aug 2008
IPC Current: C12Q000168 | C07H002102 | C12N000120 | C12N001500
US Class: 43500618 | 435006 | 4350061 | 4350691 | 4352523 | 4353201 | 5360231
Assignee Applicant: The Regents of the University of California
Title: System for protease mediated protein expression
Usefulness: System for protease mediated protein expression
Summary: The polynucleotide and polypeptide are useful for detecting, examining, and exploiting protease mediated protein expression. It can also be used to understand and characterize the physiology of proteolytic processes such as those involved in programmed cell death, including determining the role of proteases in both normal cellular processes as well as in the context of specific diseases, and in targetting therapeutics to cells.
- 主要類別
- 生物醫學
- 細分類別
- DNA /基因工程
- 申請號碼
- 7410765
- 其他
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State Of Development
The protease bioassay was tested by making a construct that detects caspase-3 activity. Caspace-3 is activated when a cell starts executing the cell death (apoptosis) program. The DNA construct was microinjected in one-cell-stage zebrafish embryos to generate transgenic fish in which apoptotic cells expressed a reporter protein (red fluorescent protein DsRed Express, in this case). Caspase-3 activity was detected in a wide variety of cells. Further, the proteolytic activity was inhibited by the addition of a caspase-3 inhibitor to the liquid medium containing the fish. These results confirmed the ability of the new protease bioassay system to function in a wide range of cell types in a vertebrate animal model, and to allow rapid, simple detection of pharmacologic modulation of the protease of interest.
Background
Proteases or enzymes with proteolytic activity are fundamental to many key biological processes such as cell growth, cell death, blood clotting, matrix remodeling and immune defense. A large number of pathogens, including viruses, bacteria and multi-cellular parasites also use proteases to infect host cells, complete their life cycle and degrade the host immune system. Proteases have also been found to play a role in the pathogenesis of hypertension, liver cirrhosis, Alzheimers disease, autoimmune diseases, rheumatoid- or osteoarthritis and cancer.
There is considerable effort to understand the role of proteases in disease and to identify therapeutic agents targeting specific protease activities. A bottleneck for high throughput drug screening, however, is now at the level of bioassays. Many compounds initially identified using in vitro assays fail in later phases of drug development because they cannot be used in a biologically relevant environment. Therefore, drug discovery researchers are now aggressively looking for high-throughput in vivo assays using cells, or, ideally, whole organisms, to screen potential drugs.What are needed are efficient bioassays to (1) determine the role of proteases in normal and diseased cellular processes and (2) screen in biologically meaningful systems for pharmacologic modulators of proteases of interest.
Additional Technologies by these Inventors
Tech ID/UC Case
20177/2005-023-0
Related Cases
2005-023-0
- 國家/地區
- 美國
