| 摘要 |
The present invention provides a method for generating polycystronic nucleic acid vectors, said method comprising the steps of a) providing a first nucleic acid vector, said first nucleic acid vector comprising: i) an origin of replication placed in front of ii) at least one first gene of interest functionally cloned within a first expression cassette, said first expression cassette comprising a promoter sequence as well as a termination sequence, said first gene of interest being located between said promoter sequence and said termination sequence, iii) a marker gene together with its termination sequence, said marker gene being situated downstream of a target sequence for a recombinase, wherein the promoter necessary for the expression of said marker gene in a host cell is absent from said first nucleic acid vector, and optionally iv) a first further marker gene which is different from the marker gene of iii), said first further marker gene being situated within said first expression cassette of ii) or within a further expression cassette, said first further marker gene allowing for the selection of cells comprising said first nucleic acid vector during the process of generating said first nucleic acid vector; b) providing a second nucleic acid vector, said second nucleic acid vector comprising i) an origin of replication placed in front of ii) at least one second gene of interest functionally cloned within a second expression cassette, said second expression cassette comprising a promoter sequence as well as a termination sequence, said second gene of interest being located between said promoter sequence and said termination sequence, iii) a promoter situated upstream of the same target sequence for a recombinase as present in front of the marker gene of iii) in the first nucleic acid vector, wherein said promoter is suitable for the expression in a host cell of said marker comprised in the first nucleic acid vector gene, and iv) optionally a second further marker gene which is different from the marker gene of iii), second further marker gene being situated within said second expression cassette of ii) or within a further expression cassette, said second further marker gene allowing for the selection of cells comprising said first nucleic acid vector during the process of generating said second nucleic acid vector; and c) contacting, under conditions suitable for a recombination to take place, a mixture of the first and the second nucleic acid vectors with a recombinase, which recombinase specifically recombines the target sequences of iii) which is situated downstream of the promoter of the second nucleic acid vector and upstream of the marker gene of the first nucleic acid vector, respectively. |
