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.

We claim:  
     
         1 . A method of phenotypic screening for perturbations of lipid and/or cholesterol metabolism in an organism, comprising: 
 (a) randomly mutagenizing at least one gene of said organism;    (b) administering a fluorescent label to said organism by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said organism under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said organism; and    (c) analyzing the pattern and/or rate of fluorescence in said organism to identify phenotypic perturbations of lipid and/or cholesterol metabolism.    
     
     
         2 . The method of  claim 1 , wherein said organism is a vertebrate.  
     
     
         3 . The method of  claim 2 , wherein said vertebrate is a mammal.  
     
     
         4 . The method of  claim 3 , wherein said mammal is a mouse.  
     
     
         5 . The method of  claim 2 , wherein said vertebrate is a teleost.  
     
     
         6 . The method of  claim 5 , wherein said teleost is a zebrafish.  
     
     
         7 . A method of phenotypic acreening for perturbations of lipid and/or cholesterol metabolism in a zebrafish, comprising: 
 (d) randomly mutagenizing at least one gene of said zebrafish;    (e) administering a fluorescent label to said zebrafish by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said zebrafish under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said zebrafish; and    (f) analyzing the pattern and/or rate of fluorescence in said zebrafish to identify phenotypic perturbations of lipid and/or cholesterol metabolism.    
     
     
         8 . 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) identifying at least one phenotypic perturbation of lipid and/or cholesterol metabolism in an organism;    (b) adminsitering a fluorescent label to said organism having said phenotypic perturbation by providing at least one fluorescent lipid to said organism under conditions that allow for uptake of said fluorescent lipid by said organism;    (c) selecting at least one agent to treat said phenotypic perturbation in said organism;    (d) administering said agent(s) to said organism under conditions that allow for uptake of said agent(s) by said organism;    (e) analyzing a change in the pattern and/or rate of fluorescence in said organism as an indication of the efficacy of said agent(s) to treat said phenotypic perturbation; and    (f) 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 organism.    
     
     
         9 . The method of  claim 8 , wherein said organism is a vertebrate.  
     
     
         10 . The method of  claim 9 , wherein said vertebrate is a mammal.  
     
     
         11 . The method of  claim 10 , wherein said mammal is a mouse.  
     
     
         12 . The method of  claim 9 , wherein said vertebrate is a teleost.  
     
     
         13 . The method of  claim 12 , wherein said teleost is a zebrafish.  
     
     
         14 . 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: 
 (g) identifying at least one phenotypic perturbation of lipid and/or cholesterol metabolism in a zebrafish;    (h) administering a fluorescent label to said zebrafish having said phenotypic perturbation by providing at least one fluorescent lipid to sadi zebrafish under conditions that allow for uptake of said fluorescent lipid by said zebrafish;    (i) selecting at least one agent to treat said phenotypic perturbation in said organism;    (j) administering said agent(s) to said zebrafish under conditions that allow for uptake of said agent(s) by said zebrafish;    (k) analyzing a change in the pattern and/or rate of fluorescence in said zebrafish as an indication of the efficacy of said agent(s) to treat said phenotypic perturbation; and    (l) 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 zebrafish.    
     
     
         15 . A method of identifying a gene(s) involved in the regulation of lipid and/or cholesterol processing, comprising: 
 (a) mutagenizing at least one organism by targeting a specific genetic site or DNA sequence with unknown function;    (b) administering a fluorescent label to said mutagenized organism by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said mutagenized organism under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said mutagenized organism;    (c) comparing the pattern and/or rate of fluorescence in said mutagenized organisms to the pattern and/or rate of fluorescence observed in an unmutagenized organism of the same species as the mutagenized organism, which unmutagenized organism was administered a fluorescent label by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said unmutagenized organism under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said unmutagenized organism; and identifying a gene(s) involved in the regulation of lipid and/or cholesterol processing by a change in the pattern and/or rate of fluorescence in said mutagenized organism.    
     
     
         16 . The method of  claim 15 , wherein said organism is a vertebrate.  
     
     
         17 . The method of  claim 16 , wherein said vertebrate is a mammal.  
     
     
         18 . The method of  claim 17 , wherein said mammal is a mouse.  
     
     
         19 . The method of  claim 16 , wherein said vertebrate is a teleost.  
     
     
         20 . The method of  claim 19 , wherein said teleost is a zebrafish.  
     
     
         21 . A method of identifying a gene(s) involved in the regulation of lipid and/or cholesterol processing, comprising: 
 (a) mutagenizing at least one zebrafish by targeting a specific genetic site or DNA sequence with unknown function in said zebrafish;    (b) administering a fluorescent label to said mutagenized zebrafish by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said mutagenized zebrafish under conditions that allow for uptake of said quenched fluorescent phospholipid and/or NBD cholesterol by said mutagenized zebrafish;    (c) comparing the pattern and/or rate of fluorescence in said mutagenized zebrafish to the pattern and/or rate of fluorescence observed in an unmutagenized zebrafish, which unmutagenized zebrafish was administered a fluorescent label by providing at least one quenched fluorescent phospholipid analogue and/or NBD cholesterol to said unmutagenized zebrafish under conditions that allow for uptake of said quenced fluorescent phospholipid and/or NBD cholesterol by said unmutagenized zebrafish; and    (d) identifying a gene(s) involved in the regulation of lipid and/or cholesterol processing by a change in the pattern and/or rate of fluorescence in said mutagenized zebrafish.