Cholesterol 7 .alpha.-hydroxdylase gene regulatory elements and transcription factors
AssigneeNortheastern Ohio University
InventorJohn Young Ling Chiang
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Abstract
DNA regulatory elements that control cholesterol 7.alpha.-hydroxylase expression are disclosed, including bile acid responsive elements. A gene construct comprising at least one CYP7 regulatory element and a reporter gene is used to transfect HepG2 cells. Confluent transfected HepG2 cells are employed in an assay to detect a compound that modulates cholesterol 7.alpha.-hydroxylase enzyme regulation. A method for screening compounds that inhibit or stimulate expression of the enzyme is provided, as well as a method for detecting and isolating transcription factors of the cholesterol 7.alpha.-hydroxylase gene. A transcription factor of 57 KDa is identified which is useful in an assay for determining regulation of CYP7 expression.
Claims26
A culture of HepG2 cells transfected with CYP7 DNA that is regulatory with respect to CYP7 expression in the presence of bile acids, wherein
- (a) said culture is confluent, and
- (b) said culture is responsive to the presence of bile acids.
A method for determining an agent's effect on CYP7 DNA comprising the steps of:
- (a) bringing into contact with an agent, a culture of HepG2 cells transfected with CYP7 DNA that is regulatory with respect to bile acids, wherein
- (i) said culture is confluent, and
- (ii) said culture is responsive to the presence of bile acids, and
- (b) determining whether said agent inhibits or stimulates the regulatory activity of said CYP7 DNA.
A method of obtaining a culture of HepG2 cells responsive to the presence of bile acids, comprising:
- (a) transfecting a culture of HepG2 cells with CYP7 DNA having a bile acid responsive element;
- and
- (b) culturing said culture to substantially complete confluency such that said culture is capable of responding to the presence of bile acids.
Dependencies are inferred from the claim text.