Temperature-dependent insertion of genetic material into genomic DNA

A method for insertion of genetic material into genomic DNA comprising

• (a) incubating host cells comprising recipient viral vector and donor nucleic acid vector under conditions and for a time suitable for promoting recombination between the recipient viral vector and the donor nucleic acid vector, wherein
• (i) the recipient viral vector comprises
• (A) a gene encoding a toxic protein, wherein expression of the toxic protein is temperature sensitive;
• or the toxic protein itself is only toxic at a particular temperature;
• and
• (B) two recombination sites flanking the gene encoding the toxic protein, wherein the two recombination sites are unique in the recipient viral vector;
• wherein
• (ii) the donor nucleic acid vector comprises
• (A) a donor gene;
• and
• (B) two recombination sites flanking the donor gene, wherein the two recombination sites are (I) unique in the donor nucleic acid vector, and (II) identical to the two recombination sites in the recipient viral vector;
• and wherein
• (iii) the host cell comprises a promiscuous DNA polymerase capable of synthesizing recipient viral vector DNA, and
• wherein the conditions comprise incubating the host cells at a first temperature at which the toxic protein is not expressed;
• (b) subsequently culturing the host cells at a second temperature at which the toxic protein is expressed by the recipient viral vector;
• and
• (c) selecting viruses that grow at the second temperature, wherein viruses that grow at the second temperature are recombinant recipient viral vectors in which the donor gene has been inserted into the recipient viral vector, replacing the toxic gene.