Inducible site-specific recombination for the activation and removal of transgenes in transgenic plants

Abstract

Disclosed is an inducible promoter system in conjunction with a site-specific recombination system which allows (i) specific activation of transgenes at specific times or (ii) excision and removal of transgenes (e.g., antibiotic resistance markers) from transgenic plants. These “suicide” gene cassettes, including the recombination system itself, can be evicted from the plant genome once their function has been exerted. The system is based on the ability to temporally and spatially induce the expression of CRE recombinase which then binds to directly repeated lox sites flanking the transgene in question leading to the precise excision of the gene cassette. Also disclosed is a method to activate an inverted, and therefore silent, transgene by placing two lox sites in opposite orientations flanking the transgene. This results in inversion of the intervening DNA fragment in the presence of CRE recombinase. This activation can be timed by placing the CRE recombinase under the control of an inducible promoter.

What is claimed is:  
     
         1 . A plant transformation vector comprising a gene encoding a transcription factor, an inducible gene, a promoter, a gene of interest, and a pair of recombination sites flanking a terminator.  
     
     
         2 . The vector of  claim 1 , wherein the transcription factor together with an inducer induces expression of the inducible gene.  
     
     
         3 . The vector of  claim 1 , wherein the inducible gene is under control of a vertebrate hormone inducible promoter system.  
     
     
         4 . The vector of  claim 3 , wherein the hormone inducible promoter system is the GVG inducible promoter system.  
     
     
         5 . The vector of  claim 1 , wherein the inducible gene encodes a recombinase which cleaves at the recombination sites.  
     
     
         6 . The vector of  claim 5 , wherein the recombinase excises the terminator when the inducible gene is induced.  
     
     
         7 . The vector of  claim 6 , wherein the gene of interest is under the control of the promoter when the terminator is excised.  
     
     
         8 . The vector of  claim 1 , wherein the inducible gene encodes CRE, FLP, GIN or R.  
     
     
         9 . The vector of  claim 1 , wherein the recombination sites are lox, FRT, gix or RS.  
     
     
         10 . The vector of  claim 1 , wherein the gene of interest encodes LEAFY.  
     
     
         11 . The vector of  claim 1 , wherein the inducible gene encodes a protein comprising an N-terminal transit peptide for chloroplast targeting.  
     
     
         12 . A method for expressing a gene in a transgenic plant or plant cell at a specific time comprising: 
 a) transfecting said plant or plant cell with the vector of  claim 1;  and    b) adding an inducer to induce the inducible gene of said vector,    wherein the inducible gene of the vector expresses a product which cleaves the terminator from the vector thereby placing the gene of interest of the vector under the control of the promoter of the vector resulting in expression of the gene of interest subsequent to addition of the inducer.    
     
     
         13 . The method of  claim 12 , wherein the inducible gene is under control of a vertebrate hormone inducible promoter system.  
     
     
         14 . The method of  claim 13 , wherein the hormone inducible promoter system is the GVG inducible promoter system.  
     
     
         15 . A plant or plant cell comprising the vector of  claim 1 .  
     
     
         16 . A plant transformation vector comprising a gene encoding a transcription factor, an inducible gene, a promoter, a gene of interest, and a pair of recombination sites flanking the gene of interest.  
     
     
         17 . The vector of  claim 16 , wherein said transcription factor together with an inducer induces expression of the inducible gene.  
     
     
         18 . The vector of  claim 16 , wherein the inducible gene is under control of a vertebrate hormone inducible promoter system.  
     
     
         19 . The vector of  claim 18 , wherein the hormone inducible promoter system is the GVG inducible promoter system.  
     
     
         20 . The vector of  claim 16 , wherein the inducible encodes a recombinase which cleaves at the recombination sites.  
     
     
         21 . The vector of  claim 20 , wherein the recombinase causes inversion of the gene of interest.  
     
     
         22 . The vector of  claim 21 , wherein upon inversion of the gene of interest, said gene of interest is under the control of the promoter.  
     
     
         23 . The vector of  claim 16 , wherein the inducible gene encodes CRE, FLP, GIN or R.  
     
     
         24 . The vector of  claim 16 , wherein the recombination sites are lox, FRT, gix or RS.  
     
     
         25 . The vector of  claim 16 , wherein the gene of interest encodes LEAFY.  
     
     
         26 . The vector of  claim 16 , wherein the inducible gene encodes a protein comprising an N-terminal transit peptide for chloroplast targeting.  
     
     
         27 . A method for expressing a gene in a transgenic plant or plant cell at a specific time comprising: 
 a) transfecting said plant or plant cell with the vector of  claim 16;  and    b) adding an inducer to induce the inducible gene of the vector,    wherein the inducible gene of the vector expresses a product which causes inversion of the gene of interest of the vector thereby placing the gene of interest under the control of the promoter of the vector resulting in expression of the gene of interest subsequent to addition of the inducer.    
     
     
         28 . The method of  claim 27 , wherein the inducible gene is under control of a vertebrate hormone inducible promoter system.  
     
     
         29 . The method of  claim 28 , wherein the hormone inducible promoter system is the GVG inducible promoter system.  
     
     
         30 . A plant or plant cell comprising the vector of  claim 16 .  
     
     
         31 . A plant transformation vector comprising an inducible gene, a promoter, a gene of interest and a pair of recombination sites flanking a terminator.  
     
     
         32 . A plant transformation vector comprising an inducible gene, a promoter, a gene of interest and a pair of recombination sites flanking the gene of interest.  
     
     
         33 . A plant transformation vector comprising an inducible gene, a marker gene, a gene of interest and a pair of recombination sites flanking the inducible gene and the marker gene.