Optimized t-dna transfer and vectors therefor

Abstract

The present invention relates to T-DNA vectors and methods for obtaining transgenic eukaryotes using said vectors. The transgenic eukaryotes are characterized in that they contain the T-DNA but not the illegitimately transferred vector backbone sequence. This is achieved by modifying the T-DNA borders such that they are more efficiently nicked or such that they allow elimination of illegitimately transferred vector backbone sequences by means of recombination.

1 . A T-DNA transformation vector comprising T-DNA with flanking T-DNA borders, characterized in that said T-DNA borders are modified resulting in preventing the integration of vector backbone sequences in the transformed cells and/or in curing transformed cells of integrated vector backbone sequences. 
     
     
         2 . The vector of  claim 1  wherein said modifications include a modification of the right T-DNA border comprising a single right border core sequence flanked by a right border outer region and/or a modification, including multiplication, of the left T-DNA border or part thereof. 
     
     
         3 . The vector of  claim 2  wherein said modification of said left T-DNA border results in efficient nicking at the left border core repeat by the nicking complex at least involving the VirD1 and/or VirD2 proteins. 
     
     
         4 . The vector of  claim 1  wherein said modified left T-DNA border comprises a single left border core sequence that is flanked by a natural left border outer region and an intra-T-DNA left border proximal region with a preferable length of 10 to 100 bp, preferably of 20-100 bp and which is enriched in the number of A- and T-residues, the percentage of AT-residues being 65 to 80%. 
     
     
         5 . The vector of  claim 1  wherein said modified left T-DNA border comprises a single left border core sequence that is flanked only by a natural left border inner region. 
     
     
         6 . The vector of  claim 1  wherein said modified left T-DNA border comprises only a single left border core sequence. 
     
     
         7 . The vector of  claim 1  wherein said modified left T-DNA border comprises the inclusion of at least two left border core sequences separated by a sequence of at least 10-20 by optionally carrying stop codons in the three reading frames and in both directions; and wherein said tandem repeat of the left border core sequences is flanked only by a left border outer region. 
     
     
         8 . The vector of  claim 1  wherein said modified left T-DNA border comprises the inclusion of at least one integral nopaline-type left border region adjacent to and downstream or upstream of the integral octopine-type left border region. 
     
     
         9 . The vector of  claim 1 , characterized in that said modifications of the T-DNA borders comprise the addition of recombination sites downstream of the left border core sequence and the addition of recombination sites upstream of the right border core sequence, said recombination sites being organized as repeats. 
     
     
         10 . The vector of  claim 9 , further comprising a second copy of a left border region upstream of and preferably adjacent to the single right border outer region and said recombination site upstream of the core sequence of said second left border region. 
     
     
         11 . A vector of  claim 9 , further comprising a recombinase gene located downstream of said recombination site downstream of said left border core sequence, and preferably, when present, adjacent to and downstream of the left border outer region. 
     
     
         12 . The vector of  claim 11 , comprising said recombinase gene flanked by repeats of recombination sites and said vector further comprising a second copy of a left border region located downstream of said recombinase gene flanked by said recombination sites. 
     
     
         13 . The vector of  claim 12 , further comprising additional recombination sites organized as repeats downstream of the second left border core sequence and upstream of the single right border core sequence. 
     
     
         14 . The vector of  claim 9 , characterized in that said recombination sites are located adjacent to and downstream and/or upstream of the left- and/or right border core sequences or are separated downstream and/or upstream from the left- and/or right border core sequences by a sequence of at least 10-20 bp in length optionally carrying stop codons in the three reading frames and in both directions. 
     
     
         15 . The vector of  claim 9 , wherein said recombination sites organized as repeats are defined as either site-specific recombination sites organized as direct repeats or as transposon border sequences organized as inverted repeats; and wherein said recombinase gene is defined as either a site-specific recombinase or a transposase gene, respectively. 
     
     
         16 . A vector for  Agrobacterium -mediated transformation, for agrolistic transformation of for gene therapy purposes comprising at least one modified T-DNA border as defined in  claim 1 , wherein said vector is chosen from binary transformation vectors, super-binary transformation vectors, co-integrate transformation vectors, Ri-derived transformation vectors and T-DNA carrying vectors used in agrolistic transformation or gene therapy. 
     
     
         17 . A method for obtaining transgenic plants, yeasts, moulds or filamentous fungi consisting of an  Agrobacterium -mediated transformation of said plants, yeasts, moulds or filamentous fungi with the transformation vector of  claim 1 . 
     
     
         18 . A method for obtaining transgenic plants, yeasts, moulds or filamentous fungi consisting of an  Agrobacterium -mediated transformation of said plants, yeasts, moulds or filamentous fungi with a transformation vector defined in  claim 9  in combination with the supply of a recombinase or transposase, for curing said resulting transformed cells from integrated vector backbone sequences possibly originating from said vector. 
     
     
         19 . A method for obtaining transgenic plants, yeasts, moulds or filamentous fungi consisting of an  Agrobacterium -mediated transformation of said plants, yeasts, moulds or filamentous fungi with a transformation vector defined in  claim 11  for curing said resulting transformed cells from integrated vector backbone sequences possibly originating from said vector, optionally in combination with the supply of a recombinase or transposase. 
     
     
         20 . A method for preventing the integration of vector backbone sequences in an  Agrobacterium -mediated transformed cell comprising enhancing the efficiency of the nicking at the left border core sequence of a T-DNA vector, including the vector according to  claim 1 , by increasing the production of the VirD1 and/or VirD2 proteins comprised within the T-DNA nicking complex. 
     
     
         21 . The method of  claim 20 , involving the integration of at least one additional copy of the virD locus into the genome or into an extrachromosomal entity of  Agrobacterium.    
     
     
         22 . The method of  claim 21 , wherein said additional copy of the virD locus is selected from the octopine-type virD locus or the nopaline-type virD locus. 
     
     
         23 . The method of  claim 17 , further comprising increasing the production of VirD1 and/or VirD2 proteins comprised within the T-DNA nicking complex. 
     
     
         24 . The method of agrolistic-based transformation of a eukaryotic cell using a T-DNA vector modified according to the alterations as defined in the vectors of  claim 1 . 
     
     
         25 . A gene therapy method comprising any use of a T-DNA vector modified according to the alterations as defined in the vector of  claim 1 . 
     
     
         26 . A recombinant host cell containing the vector as defined in  claim 1 . 
     
     
         27 . The host cell of  claim 26  identified as being an  Agrobacterium tumefaciens.    
     
     
         28 . A transgenic plant cell or plant obtainable by an  Agrobacterium -mediated transformation method according to  claim 17 , part thereof, or progeny thereof. 
     
     
         29 . A transgenic yeast, mould or filamentous fungus obtainable by an  Agrobacterium -mediated transformation method according to  claim 17 .