In vivo method for identifying target sites for antisense-mediated inhibition of a selected gene

Abstract

A method for making a directed antisense library against a target transcript is described. A cDNA of the target transcript is cloned in an appropriate cloning vector. Next, a plurality of deletion derivatives of the cloned cDNA is prepared such that the deletions serially extend into the cDNA from one end thereof. The resulting deletion library is then treated such that cDNA is removed from the other end of each cDNA insert, thus obtaining a fragment library having fragments of a selected size. An antisense gene is then inserted into each fragment of the fragment library, resulting in the directed antisense library. An illustrative antisense gene in the hammerhead ribozyme catalytic core. Plasmids for making the antisense library, plasmids and methods for making the fragment library, and a method for identifying target sites for antisense-mediated gene inhibition are also described.

The subject matter claimed is:  
     
         1 . A method for identifying target sites for antisense-mediated inhibition of a selected gene comprising: 
 (a) constructing a directed antisense library targeted at the selected gene wherein the library is contained in a cloning vector having a promoter configured for transcribing antisense transcripts from the directed antisense library in suitable cells wherein the selected gene is expressed as a target transcript;    (b) transforming a plurality of the suitable cells such that each of the plurality of suitable cells transcribes an antisense transcript that has access to the target transcript for potential inactivation thereof;    (c) identifying a cell wherein an antisense transcript inactivates the target transcript; and    (d) analyzing the antisense transcript that inactivates the target transcript and determining a target site on the antisense transcript that is associated with inactivation of the target transcript.    
     
     
         2 . The method of  claim 1  wherein constructing a directed antisense library targeted at the selected gene comprises: 
 (a) preparing a double-stranded cDNA, comprising a first end, a second end, and a central site thereof, from the target transcript and cloning the cDNA in a cloning vector comprising a promoter configured such that an antisense transcript of the cDNA is synthesized upon transcription mediated by the promoter, resulting in a cloned cDNA;  
 (b) creating a plurality of deletion derivatives of the cloned cDNA wherein each of the plurality of deletion derivatives has a deletion extending from the first end into the cloned cDNA such that the plurality of deletion derivatives comprises a deletion library comprising deletions extending serially into the cDNA;  
 (c) reducing the size of the cDNA contained in the deletion library to a preselected size by removing a portion of the cDNA from the second end thereof to result in a fragment library;  
 (d) inserting an antisense gene DNA into the central site of the cDNA in the fragment library, thereby obtaining the antisense library.  
 
     
     
         3 . The method of  claim 2  wherein said plurality of deletion derivatives is created with exonuclease III resection of the cloned cDNA.  
     
     
         4 . The method of  claim 2  wherein the reducing the size of the cDNA contained in the deletion library to a preselected size comprises digesting the deletion library with a type IIS restriction endonuclease.  
     
     
         5 . The method of  claim 2  wherein the inserting an antisense gene DNA into the central site of the cDNA in the fragment library comprises digesting the fragment library with a type IIS restriction endonuclease, thereby creating the central site, and ligating the antisense gene DNA at the central site.  
     
     
         6 . The method of  claim 2  wherein the antisense gene DNA comprises a ribozyme catalytic core.  
     
     
         7 . The method of  claim 2  wherein the ribozyme catalytic core is a hammerhead ribozyme catalytic core.  
     
     
         8 . The method of  claim 1  wherein the cloning vector is pShuttle (SEQ ID NO:14).  
     
     
         9 . The method of  claim 1  wherein the cloning vector is pBK (SEQ ID NO:17).