Claims
- 1. A retroviral packaging cell comprising a suitable mammalian cell having therein (i) a first recombinant nucleic acid comprising MMLV gag and pol genes and a selectable marker, and (ii) a second recombinant nucleic acid comprising RD114 envelope gene and a selectable marker, wherein the MMLV gag and pol genes and the RD114 envelope gene are stably expressed in the cell.
- 2. The cell of claim 1, wherein the suitable mammalian cell is a mouse 3T3 cell.
- 3. The cell of claim 1, wherein the suitable mammalian cell is a human 293 cell.
- 4. The cell of claim 1, wherein each of the first and second nucleic acids is DNA.
- 5. The retroviral packaging cell designated RDF 21 (ATCC Patent Deposit Designation PTA-4440).
- 6. A method for producing a retroviral packaging cell comprising:
(a) transfecting a plurality of suitable mammalian cells with a recombinant nucleic acid comprising RD114 envelope gene and a selectable marker, each mammalian cell having therein a recombinant nucleic acid comprising MMLV gag and pol genes, which gag and pol genes are stably expressed in the cell; and (b) isolating from among the resulting transfected cells of step (a) a cell which stably expresses the RD114 envelope gene, thereby producing a retroviral packaging cell.
- 7. The method of claim 6, wherein the suitable mammalian cell is a GP101 cell.
- 8. A method for producing a retroviral packaging cell comprising:
(a) transfecting a plurality of suitable mammalian cells with (i) a first recombinant nucleic acid comprising the MMLV gag and pol genes and a selectable marker, and (ii) a second recombinant nucleic acid comprising RD114 envelope gene and a selectable marker; and (b) isolating from among the resulting transfected cells of step (a) a cell which stably expresses the gag, pol and RD114 genes, thereby producing a retroviral packaging cell.
- 9. The method of claim 8, wherein the suitable mammalian cell is a mouse 3T3 cell.
- 10. The method of claim 8, wherein the suitable mammalian cell is a human 293 cell.
- 11. A method for producing a retroviral producer cell comprising transducing the retroviral packaging cell of claim 1 with a retroviral vector comprising a nucleic acid sequence of interest so as to cause the cell to secrete virions comprising the nucleic acid sequence of interest, thereby producing a retroviral producer cell.
- 12. The method of claim 11, wherein the retroviral vector is a MMLV vector.
- 13. The method of claim 11, wherein the retroviral vector is a lentiviral vector.
- 14. A method for producing a retroviral producer cell comprising transducing the retroviral packaging cell of claim 5 with a retroviral vector comprising a nucleic acid sequence of interest so as to cause the cell to secrete virions comprising the nucleic acid sequence of interest, thereby producing a retroviral producer cell.
- 15. The method of claim 11 or 14, wherein the nucleic acid of interest comprises a therapeutic gene.
- 16. The method of claim 15, wherein the nucleic acid of interest further comprises a selectable marker.
- 17. The retroviral producer cell produced by the method of claim 11.
- 18. A method for producing high-titer retroviral supernatant comprising culturing the retroviral producer cell of claim 17 under suitable conditions, thereby producing high-titer retroviral supernatant.
- 19. The method of claim 18, further comprising the steps of collecting and concentrating the retroviral supernatant.
- 20. The retroviral producer cell produced by the method of claim 14.
- 21. A method for producing high-titer retroviral supernatant comprising culturing the retroviral producer cell of claim 20 under suitable conditions, thereby producing high-titer retroviral supernatant.
- 22. The method of claim 21, further comprising the steps of collecting and concentrating the retroviral supernatant.
- 23. The virion produced by the retroviral producer cell of claim 17.
- 24. The virion produced by the retroviral producer cell of claim 20.
- 25. A kit comprising the retroviral packaging cell of claim 1, and instructions for use.
- 26. A kit comprising the retroviral packaging cell of claim 1, and a retroviral vector comprising a nucleic acid of interest.
- 27. The kit of claim 26, further comprising instructions for use.
- 28. A kit comprising the retroviral packaging cell of claim 5, and instructions for use.
- 29. A kit comprising the retroviral packaging cell of claim 5, and a retroviral vector comprising a nucleic acid of interest.
- 30. The kit of claim 29, further comprising instructions for use.
Government Interests
[0001] This invention was made with funding from the United States National Institute of Health, Grant No. 1-RO1—HL-59887-05. Accordingly, the United States Government has certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60424077 |
Nov 2002 |
US |