Northeastern Ohio University

John Young Chiang

Diane Stroup

Genomic DNA of cholesterol 7.alpha.-hydroxylase and a minigene are disclosed. The minigene is used for making a transgenic animal that produces functionally active cholesterol 7.alpha.-hydroxylase and functions as a disease model. A cholesterol 7.alpha.-hydroxylase promoter region and reporter gene construct is provided, as well as a transgenic animal that expresses the promoter/reporter gene.

Genomic DNA of human cholesterol 7.alpha.-hydroxylase and methods for

DNA regulatory elements that control cholesterol 7.alpha.-hydroxylase (CYP7) expression are disclosed, including a CYP7 minigene that comprises nucleotides downstream from about -371 of the proximal promoter region through about the middle of intron II, inclusive of intron I. This minigene further is stably transfected into a cell line, along with a reporter gene. A method is provided for screening agents that inhibit or stimulate expression of the minigene.

Assay for agents that affect cholesterol 7alpha-hydroxylase expression

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.

Cholesterol 7 .alpha.-hydroxdylase gene regulatory elements and

Transgenic mice expressing human cholesterol 7.alpha.hydroxylase

DNA regulatory elements that control cholesterol 7.alpha.-hydroxylase expression are disclosed. A gene construct is provided comprising at least one regulatory element and a reporter gene is used in an assay to detect a compound that modulates cholesterol 7.alpha.-hydroxylase enzyme regulation. Thus, a method for screening compounds that inhibit or stimulate expression of the enzyme is provided, as well as a method for detecting and isolating the transcription factors of the cholesterol 7.alpha.-hydroxylase gene.

Cholesterol 7.alpha.-hydroxylase gene regulatory elements and methods

A catalytically active, truncated human cholesterol 7.alpha.-hydroxylase (CYP7) is provided. The truncated human CYP7 can be made recombinantly and thus recovered in relatively large amounts. Vectors and host cells for recombinant expression are provided, as well as an antibody which specifically recognizes an epitope of catalytically active, truncated human CYP7. Also provided is a method for screening a compound for its effect on expression of non-truncated human CYP7, and a method for screening a compound for its effect on non-truncated human CYP7 enzyme activity.

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Stow, OH, United States of America

John Young Chiang