High efficiency FLP site-specific recombination in mammalian cells using an optimized FLP gene
View Patent ↗The present invention provides an optimized FLP site-specific recombinase coding sequence and methods for its use. This genetically engineered FLP gene displays a marked increase in recombination efficiency compared to the native FLP gene and is therefore useful in a wide array of molecular applications.
1. A nucleic acid molecule comprising an optimized FLP recombinase coding sequence having the sequence of SEQ ID NO:1 that provides enhanced recombinase activity in a cell comprising and expressing the nucleic acid molecule.
2. An expression construct comprising the nucleic acid molecule comprising the optimized FLP recombinase coding sequence of claim 1 .
3. A method of preparing isolated cells comprising an optimized FLP recombinase coding sequence having the sequence of SEQ ID NO:1 that provides enhanced recombinase activity in a cell comprising and expressing the nucleic acid molecule, the method comprising transforming an isolated cell with the expression construct of claim 2 .
4. The method of claim 3 , wherein the isolated cells are eukaryotic cells.
5. The method of claim 4 , wherein the isolated cells are invertebrate cells.
6. The method of claim 4 , wherein the isolated cells are vertebrate cells.
7. The method of claim 6 , wherein the isolated cells are mammalian cells.
8. The method of claim 7 , wherein the isolated cells are mouse cells.
9. The method of claim 8 , wherein the isolated mouse cells are mouse ES cells.
10. An isolated cell comprising the nucleic acid molecule comprising the optimized FLP recombinase coding sequence of claim 1 .
11. The isolated cell of claim 10 , wherein the isolated cell is a eukaryotic cell.
12. The isolated cell of claim 11 , wherein the isolated cell is an invertebrate cell.
13. The isolated cell of claim 11 , wherein the isolated cell is a vertebrate cell.
14. The isolated cell of claim 13 , wherein the isolated cell is a mammalian cell.
15. The isolated cell of claim 14 , wherein the isolated cell is a mouse cell.
16. The isolated mouse cell of claim 15 , wherein the mouse cell is a mouse embryonic stem (ES) cell.
17. A nucleic acid molecule comprising an optimized FLP recombinase coding sequence having at least about 90% sequence identity to the sequence of SEQ ID NO:1 that provides enhanced recombinase activity in a cell comprising and expressing the nucleic acid molecule.
18. An expression construct comprising the nucleic acid molecule comprising the optimized FLP recombinase coding sequence of claim 17 .
19. A method of preparing isolated cells comprising an optimized FLP recombinase coding sequence having at least about 90% sequence identity to the sequence of SEQ ID NO:1 that provides enhanced recombinase activity in a cell comprising and expressing the nucleic acid molecule, the method comprising transforming an isolated cell with the expression construct of claim 18 .
20. The method of claim 19 , wherein the isolated cells are eukaryotic cells.
21. The method of claim 20 , wherein the isolated cells are invertebrate cells.
22. The method of claim 20 , wherein the isolated cells are vertebrate cells.
23. The method of claim 22 , wherein the isolated cells are mammalian cells.
24. The method of claim 23 , wherein the isolated cells are mouse cells.
25. The method of claim 24 , wherein the isolated mouse cells are mouse ES cells.
26. An isolated cell comprising the nucleic acid molecule comprising the optimized FLP recombinase coding sequence of claim 17 .
27. The isolated cell of claim 26 , wherein the isolated cell is a eukaryotic cell.
28. The isolated cell of claim 27 , wherein the isolated cell is an invertebrate cell.
29. The isolated cell of claim 27 , wherein the isolated cell is a vertebrate cell.
30. The isolated cell of claim 29 , wherein the isolated cell is a mammalian cell.
31. The isolated cell of claim 30 , wherein the isolated cell is a mouse cell.
32. The isolated cell of claim 31 , wherein the mouse cell is a mouse ES cell.