Perlecan transgenic animals and methods of identifying compounds for the treatment of amyloidoses

Abstract

The invention provides a transgenic non-human animal expressing a perlecan encoding transgene. Also provided is a double-transgenic non-human animal expressing a perlecan and an amyloid encoding transgene. A method of screening for a compound which alters the rate or extent of amyloid deposition is additionally provided. The method consists of: (a) constructing a perlecan transgenic animal; (b) administering an effective amount of a test compound to said perlecan transgenic animal; and (c) determining whether said test compound alters the extent or rate of amyloid deposition. Finally, the invention provides a method of screening for a compound which alters the rate or extent of amyloid deposition. The method consists of: (a) constructing a perlecan/amyloid double-transgenic animal; (b) administering an effective amount of a test compound to said perlecan/amyloid double-transgenic animal; and (c) determining whether said test compound alters the extent o rate of amyloid deposition.

We claim:  
     
         1 . A method for making a transgenic non-human mammal, the method comprising the steps of selecting appropriate restriction sites for ligation of cDNA clones, and ligating together 7 overlapping cDNA clones to produce a ligated cDNA clone that encodes for perlecan's ˜400 kilodalton core protein.  
     
     
         2 . The method of  claim 1  wherein the non-human mammal is a mouse.  
     
     
         3 . The method of  claim 1  further comprising the following steps: 
 a) constructing a plasmid (p Front End) containing cDNA for perlecan domains I, II and III from clone 19-J (-XbaI), clone 54 and clone DIII, by isolating the 3.3 kilobase SalI/XbaI fragment isolated from clone DIII, isolating the 1.4 kilobase Not I/Bcl I DNA fragment from clone 19-J (XbaI) and cloning it into the NotI and Bcl sites of clone 54 to produce clone 19-J/54, isolating the 1.8 kilobase NotI/Sal I DNA fragment from clone 19-J/54 and ligating it onto the Sal I site of the 3.3 kilobase SalI/XbaI fragment isolated from clone DIII, and cloning a resulting 5.1 kilobase Not I/Xba I fragment into the Not I and Xba I site of pBluescript I to produce clone pFront End;  
 b) constructing a plasmid (p Back End) containing cDNA for most of perlecan domain IV and all of domain V from clone DV and clone 12, to introduce a Hind III site into clone DV by replacing the XhoI site in clone DV with a Hind III site using Hind III linkers to produce clone DV with a Hind III site, and isolating the 3.2 kilobase Xba I/Cla I fragment from clone 12 and cloning it into the Xba I/Cla I sites of clone DV with a Hind III site from which a smaller Xba I/Cla I fragment is previously removed to produce clone p Back End;  
 c) constructing a clone pF+ B by connecting together the p Front End and p Back End plasmids from step a) and step b), and cloning the 5.1 kilobase NotI/Xba I fragment from step a) into the same restriction enzyme sites of p Back End so that clone pF+B contains cDNA for the entire core protein of perlecan except for a small region in the N-terminal part of domain IV;  
 d) constructing a plasmid (p Missing Link) containing cDNA for small region in the N-terminal part of perlecan domain IV from clone 72 and clone 7 by isolating a 1.5 kilobase NotI/Hind III fragment isolated from clone 72 and a 2.2 kilobase Hind III/Bam HI fragment from clone 7 and cloning them respectively into the NotI and Bam HI sites of pBluescript I to produce clone 72/7, and isolating a 190 base pair Ppu MI fragment from clone 7 and inserting it into a restriction enzyme site in clone 72/7 from which smaller Ppu MI fragments have been previously removed;  
 e) inserting the cDNA of p Missing Link into pF+B, isolating a 2.8 kilobase Nru I/Bss HII fragment from p Missing Link and cloning it into the NFu I and Bss HII sites of pF+ B from which a small Nru I/Bss HII fragment has been removed to produce plasmid pBS DI-V.  
 
     
     
         4 . A transgenic non-human mammal expressing a perlecan transgene, wherein the perlecan transgene comprises one or more sequences each coding for one or more fragments of perlecan, and wherein the perlecan fragments are each homologous to a portion of a perlecan domain selected from the group of perlecan domains consisting of domains I-V and splice variants of domains I-V.  
     
     
         5 . The transgenic mammal of  claim 4 , wherein the non-human mammal is a mouse.  
     
     
         6 . A method for making a transfected cell, the method comprising the steps of selecting appropriate restriction sites for ligation of cDNA clones, and ligating together 7 overlapping cDNA clones to produce a ligated cDNA clone in the transfected cell that encodes for perlecan's ˜400 kilodalton core protein.  
     
     
         7 . The method of  claim 6  wherein the transfected cell encodes for a perlecan selected from the group of perlecans consisting of mouse perlecan, bovine perlecan and human perlecan.  
     
     
         8 . The method of  claim 6  wherein the cell type selected for transfection is selected from the group of cell types consisting of COS cells and P19 cells.  
     
     
         9 . A transfected cell expressing a perlecan encoding transgene, wherein the perlecan transgene comprises one or more sequences each coding for one or more fragments of perlecan, and wherein the perlecan fragments are each homologous to a portion of a perlecan domain selected from the group of perlecan domains consisting of domains I-V and splice variants of domains I-V.  
     
     
         10 . A double-transgenic non-human animal expressing a perlecan and an amyloid encoding transgene.  
     
     
         11 . A method of screening for a compounds which alters the rate or extent of amyloid deposition comprising: 
 (a) constructing a perlecan transgenic animals;    (b) administering an effective amount of a test compounds to said perlecan transgenic animal; and    (c) determining whether said test compound alters the extent or rate of amyloid deposition.    
     
     
         12 . A method of screening for a compound which alters the rate or extent of amyloid deposition comprising: 
 (a) constructing a perlecan/amyloid double-transgenic animal;    (b) administering an effective amount of a test compound to said perlecan/amyloid double-transgenic animal; and    (c) determining whether said test compound alters the extent or rate of amyloid deposition.    
     
     
         13 . A method for making a transgenic non-human mammal using unique restriction sites to ligate together 7 overlapping cDNA clones to produce a single ˜12 kb cDNA clone that encodes for perlecan's ˜400 kilodalton core protein.  
     
     
         14 . The transgenic non-human mammal of  claim 7 , wherein said neuropathological condition is selected from the group consisting of an Alzheimer's disease, an Alzheimer's-like disease, a familial Alzheimer's disease, and a hereditary cerebral hemorrhage with amyloidosis-Dutch type.