# US 2014/0193859 A1 · Temperature-dependent insertion of genetic material into genomic DNA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0193859 A1 - Document ID: US20140193859A1 - Status: Published application - Kind: A1 - Application number: 14/149,398 - Filed: 2014-01-07 - Published: 2014-07-10 - Earliest claimed: 2013-01-08 - Inventors: Bertram JACOBS; Karen KIBLER - Web page: https://patentgrep.com/patent/US20140193859A1 ## CPC - C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease - C12N 15/86: Viral vectors - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - C12N 2710/24143: viral genome or elements thereof as genetic vector - C12N 2800/40: Systems of functionally co-operating vectors - C12N 2800/50: Vectors for producing vectors - C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present invention provides improved methods and reagents for insertion of genetic material into genomic DNA. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the host cell is a eukaryotic cell. ### Claim 3 (depends on 1) The method of claim 1, wherein the host cell is a mammalian cell. ### Claim 4 (depends on 1) The method of claim 1, wherein the recipient viral vector comprises a Pox viral genome. ### Claim 5 (depends on 1) The method of claim 1, wherein the recipient viral vector comprises a vaccinia viral genome. ### Claim 6 (depends on 1) The method of claim 1, wherein the toxic protein is selected from the group consisting of protein kinase R (PKR), DNAses, RNAses, restriction endonucleases, proteases, and proteins that induce apoptosis. ### Claim 7 (depends on 1) The method of claim 1, wherein expression of the gene encoding the toxic protein is controlled by a heterologous temperature sensitive regulatory system, wherein the temperature sensitive element is present, or is encoded by a nucleic acid present, between the two recombination sites. ### Claim 8 (depends on 7) The method of claim 7, wherein the heterologous temperature sensitive regulatory system comprises a system selected from the group consisting of (i) temperature sensitive lac repressor, wherein the gene encoding the toxic protein is operatively linked to a Lac operator, (ii) temperature-sensitive λ repressor CI857, and (iii) lactic acid bacteriophage temperature-inducible gene expression systems. ### Claim 9 (depends on 1) The method of claim 1 wherein the donor nucleic acid vector comprises a donor gene encoding a therapeutic protein and/or an antigen for vaccine production. ### Claim 10 (depends on 1) The method of claim 1, wherein the DNA polymerase is a Pox virus DNA polymerase. ### Claim 11 (depends on 1) The method of claim 1, wherein the DNA polymerase is a vaccinia virus DNA polymerase. ### Claim 12 A nucleic acid cassette, comprising: (a) a gene encoding a toxic protein; (b) a regulatory element operatively linked to the gene encoding the toxic protein; and (c) a temperature sensitive repressor of the regulatory element. ### Claim 13 (depends on 12) The cassette of claim 12, wherein the toxic protein is selected from the group consisting of protein kinase R (PKR), DNAses, RNAses, restriction endonucleases, proteases, and proteins that induce apoptosis. ### Claim 14 (depends on 12) The cassette of claim 12, wherein the regulatory element comprises a lac operator and the temperature sensitive repressor comprises a temperature sensitive lac repressor ### Claim 15 A recombinant viral vector, comprising (a) a gene encoding a toxic protein, wherein expression of the toxic protein is temperature sensitive; and (b) two recombination sites flanking the gene encoding the toxic protein, wherein the two recombination sites are unique in the recipient viral vector. ### Claim 16 (depends on 15) The recombinant viral vector of claim 15, further comprising: (c) a regulatory element operatively linked to the gene encoding the toxic protein; and (d) a temperature sensitive repressor of the temperature sensitive regulatory system. ### Claim 17 (depends on 15) The recombinant viral vector of claim 15, wherein the toxic protein is selected from the group consisting of protein kinase R (PKR), DNAses, RNAses, restriction endonucleases, proteases, and proteins that induce apoptosis. ### Claim 18 (depends on 16) The recombinant viral vector of claim 16, wherein the regulatory element comprises a lac operator and the temperature sensitive repressor comprises a temperature sensitive lac repressor ### Claim 19 (depends on 15) Recombinant viral particles comprising the recombinant viral vectors of claim 15.