High throughput genetic screening of lipid and cholesterol processing using fluorescent compounds

Abstract

The present invention utilizes fluorescent lipids, particularly quenched phospholipid or cholesterol analogues, to facilitate screening for phenotypes representing perturbations of lipid processing; screening for genetic mutations that lead to disorders of phospholipid and/or cholesterol metabolism; and screening of compounds designed to treat disorders of phospholipid and/or cholesterol metabolism. The present invention is a mutant fish containing a metabolic defect affecting phospholipid and cholesterol processing. The mutant fish is useful for studying phospholipid and cholesterol metabolism and for screening compounds designed to treat disorders of phospholipid and/or cholesterol metabolism.

We claim:  
     
         1 . A mutant fish whose genome comprises a homozygous disruption in a metabolic pathway of cholesterol and/or lipid processing, wherein said disruption reduces cholesterol absorption in a gall bladder of said mutant fish and wherein said mutant fish are phenotypically normal.  
     
     
         2 . A mutant fish of  claim 1 , wherein the homozygous disruption results in a reduction in cholesterol absorption in a gall bladder of said mutant fish as compared to wild-type fish.  
     
     
         3 . A method of identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism, comprising: 
 (a) administering a fluorescent label to said mutant fish of  claim 1  by providing at least one fluorescent lipid to said mutant fish under conditions that allow for uptake of said fluorescent lipid by said mutant fish;    (b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;    (c) administering said agent(s) to said mutant fish under conditions that allow for uptake of said agent(s) by said mutant fish prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said mutant fish as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and    (e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said mutant fish.    
     
     
         4 . A method of identifying an agent(s) to reduce lipid and/or cholesterol metabolism, comprising: 
 (a) administering a fluorescent label to said mutant fish of  claim 1  by providing at least one fluorescent lipid to said mutant fish under conditions that allow for uptake of said fluorescent lipid by said mutant fish;    (b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;    (c) administering said agent(s) to said mutant fish under conditions that allow for uptake of said agent(s) by said mutant fish prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said mutant fish as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and    (e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said mutant fish.    
     
     
         5 . A method of identifying an agent(s) to enhance lipid and/or cholesterol metabolism, comprising: 
 (a) administering a fluorescent label to said mutant fish of  claim 1  by providing at least one fluorescent lipid to said mutant fish under conditions that allow for uptake of said fluorescent lipid by said mutant fish;    (b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;    (c) administering said agent(s) to said mutant fish under conditions that allow for uptake of said agent(s) by said mutant fish prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said mutant fish as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and    (e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said mutant fish.    
     
     
         6 . A method of identifying an agent(s) to reduce cholesterol absorption, comprising 
 (a) administering a fluorescent label to said mutant fish of  claim 1  by providing at least one fluorescent lipid to said mutant fish under conditions that allow for uptake of said fluorescent lipid by said mutant fish such that fluorescence is observed in a gall bladder of said mutant fish;    (b) selecting at least one agent to reduce cholesterol absorption in said mutant fish;    (c) administering said agent(s) to said mutant fish under condition that allow for uptake of said agent(s) by said mutant fish prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said mutant fish as an indication of the efficacy of said agent(s) to reduce cholesterol absorption; and    (e) identifying an agent(s) to reduce cholesterol absorption by a reduction in the pattern and/or rate of fluorescence in a gall bladder of said mutant fish.    
     
     
         7 . A method of identifying candidate functional mediators of the homozygous disruption in the mutant fish of  claim 1 , comprising 
 (a) producing an F 1  generation fish by crossing a first mutant fish whose genome comprises a heterozygous disruption in the metabolic pathway of cholesterol and/or lipid processing, wherein said disruption when homozygous reduces cholesterol absorption in a gall bladder of a fish having said homozygous disruption but produces no phenotypic change in a fish having said homozygous disruption, with a second mutant fish whose genome comprises at least one random mutation;    (b) selecting from the offspring of the cross of step (a) those fish whose genome comprises the heterozygous disruption of said first mutant fish;    (c) producing an F 2  generation fish by intercrossing the offspring selected in step (b);    (d) selecting from the offspring of the cross of step (c) those fish whose genome comprises the heterozygous disruption of the mutant fish of  claim 1;     (e) producing an F 3  generation fish by intercrossing the offspring selected in step (d);    (f) administering a fluorescent label to the selected F 3  offspring of step (d) by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said selected F 3  offspring under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said selected F 3  offspring;    (g) comparing the pattern and/or rate of fluorescence in said selected F 3  offspring to the pattern and/or rate of fluorescence observed in a wild-type fish and/or mutant fish of  claim 1 , which wild-type fish and/or mutant fish of  claim 1  was administered a fluorescent label by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said wild-type fish and/or mutant fish under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said wild-type fish and/or mutant fish of  claim 1;  and    (h) identifying candidate functional mediators of the homozygous disruption in the mutant fish of  claim 1  by a change in the pattern and/or rate of fluorescence in said selected F 3  offspring as compared to the pattern and/or rate of fluorescence in wild-type fish and/or mutant fish of  claim 1 .    
     
     
         8 . A cell line derived from the mutant fish of  claim 1 .  
     
     
         9 . The cell line of  claim 8 , wherein the cell line is derived from a liver of the mutant fish of  claim 1 .  
     
     
         10 . The cell of  claim 8 , wherein the cell line is derived from a gall bladder of the mutant fish of  claim 1 .  
     
     
         11 . A method of identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism, comprising: 
 (a) fluorescently labeling a cell isolated from at least one of the cell lines of  claim 8 ,  9 , or  10  by contacting said cell with at least one fluorescent lipid under conditions that allow for uptake of said fluorescent lipid by said cell;    (b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;    (c) contacting said agent(s) with said cell under conditions that allow for uptake of said agent(s) by said cell prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said cell as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and    (e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said cell.    
     
     
         12 . A method of identifying an agent(s) to reduce lipid and/or cholesterol metabolism, comprising: 
 (a) fluorescently labeling a cell isolated from at least one of the cell lines of  claim 8 ,  9 , or  10  by contacting said cell with at least one fluorescent lipid under conditions that allow for uptake of said fluorescent lipid by said cell;    (b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;    (c) contacting said agent(s) with said cell under conditions that allow for uptake of said agent(s) by said cell prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said cell as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and    (e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said cell.    
     
     
         13 . A method of identifying an agent(s) to enhance lipid and/or cholesterol metabolism, comprising: 
 (a) fluorescently labeling a cell isolated from at least one of the cell lines of  claim 8 ,  9 , or  10  by contacting said cell with at least one fluorescent lipid under conditions that allow for uptake of said fluorescent lipid by said cell;    (b) selecting at least one agent to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism;    (c) contacting said agent(s) with said cell under conditions that allow for uptake of said agent(s) by said cell prior to, after, or simultaneously with step (a);    (d) analyzing a change in the pattern and/or rate of fluorescence in said cell as an indication of the efficacy of said agent(s) to treat a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism; and    (e) identifying an agent(s) to treat either prophylatically or therapeutically a disease or disorder characterized by aberrant or abnormal lipid and/or cholesterol metabolism by a change in the pattern and/or rate of fluorescence in said cell.    
     
     
         14 . A method of identifying candidate functional mediators of the homozygous disruption in the mutant fish of  claim 1 , comprising 
 (a) providing a first fluorescent label with a cell isolated from at least one of the cell lines of  claim 8 ,  9 , or  10  by contacting at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol with said cell under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol to said cell;    (b) analyzing the pattern and/or rate of fluorescence of said first fluorescent label in said cell;    (c) randomly re-mutagenizing the DNA of said cell;    (d) providing a second fluorescent label to said cell by contacting at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol, wherein said second fluorescent label has a fluorescence emission profile that contrasts with the fluorescence emission profile of said first fluorescent label, with said cell under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol;    (e) comparing the pattern and/or rate of fluorescence of said second fluorescent label in said cell to the pattern and/or rate of fluorescence observed in said cell in step (b); and    (f) identifying candidate functional mediators of the homozygous disruption in the mutant fish of  claim 1  by a change in the pattern and/or rate of fluorescence in said cell.