System for producing synthetic promoters

Abstract

The present application discloses a method for making and selecting a transcription enhancing combined promoter cassette, which includes constructing a library of randomly combined transcription regulatory elements, which comprise double stranded DNA sequence elements that are recognized by transcription regulators; inserting the combined transcription regulatory elements upstream of a minimum promoter followed by a reporter gene in a vector; inserting the vector into a host cell; and then screening for the cells showing enhanced expression of the reporter gene, and identifying the combined promoter cassette in the cell.

1 . A method for making and selecting a transcription enhancing combined promoter cassette comprising: 
 (i) constructing a library of randomly combined transcription regulatory elements, each of which comprises double stranded DNA sequence elements that are recognized by a specific transcription regulator and flanking sequences;    (ii) inserting the combined transcription regulatory elements upstream of a minimum promoter followed by a reporter gene in a vector;    (iii) inserting the vector into a host cell; and    (iv) screening for the cells showing enhanced expression of the reporter gene, and identifying the combined promoter cassette in the cell.    
     
     
         2 . The method according to  claim 1 , wherein the library of randomly combined transcription regulatory elements is made by mixing individual double stranded DNA sequence elements that are recognized by transcription regulators together under ligation reaction conditions.  
     
     
         3 . The method according to  claim 1 , wherein the double stranded DNA sequence element comprises spacer nucleotides at either or both ends.  
     
     
         4 . The method according to  claim 1 , wherein the reporter gene expression is screened by fluorescence-activated cell sorter (FACS).  
     
     
         5 . The method according to  claim 1 , wherein the DNA sequence elements that are recognized by transcription regulators are obtained by DNA microarray profiling of transcription regulators and co-regulators.  
     
     
         6 . The method according to  claim 1 , wherein the reporter gene is lacZ or GFP.  
     
     
         7 . The method according to  claim 1 , wherein the host cell is cancer cell.  
     
     
         8 . The method according to  claim 7 , wherein the cancer is neuroblastoma.  
     
     
         9 . A vector comprising the combined promoter cassette according to  claim 1 .  
     
     
         10 . The vector according to  claim 9 , which is a plasmid.  
     
     
         11 . The vector according to  claim 9 , which is viral.  
     
     
         12 . The vector according to  claim 9 , which is transiently expressed.  
     
     
         13 . The vector according to  claim 9 , which is integrated into the genome of a host cell.  
     
     
         14 . A host cell comprising the vector according to  claim 9 .  
     
     
         15 . The host cell according to  claim 14 , which is a prokaryotic cell.  
     
     
         16 . The host cell according to  claim 14 , which is a eukaryotic cell.  
     
     
         17 . The host cell according to  claim 16 , which is a mammalian cell.  
     
     
         18 . The host cell according to  claim 17 , which is a human cell.  
     
     
         19 . The host cell according to  claim 18 , which is a cancer cell.