Compositions and methods for metabolic selection of transfected cells

1. A host cell transformed with a vector comprising a polynucleotide encoding a 3-ketosteroid reductase and a polynucleotide encoding a heterologous polypeptide, wherein the host cell is a eukaryotic cell and is auxotrophic for cholesterol.

2. The host cell of claim 1 , wherein said 3-ketosteroid reductase comprises a murine 3-ketosteroid reductase.

3. The host cell of claim 1 , wherein said polynucleotide encoding said reductase comprises SEQ ID NO: 1 or SEQ ID NO:2, or encodes an amino acid sequence comprising SEQ ID NO:3 or SEQ ID NO:4.

4. The host cell of claim 1 , wherein said vector is a recombinant DNA expression vector.

5. The host cell of claim 4 , wherein the recombinant DNA expression vector further comprises at least a first transcription unit under control of the human cytomegalovirus promoter.

6. The host cell of claim 1 , wherein said host cell is selected from the group consisting of NS-O, NS-I, and CHO-215.

7. The host cell of claim 1 , wherein said host cell is an NS-O mouse myeloma cell.

8. A kit comprising:

a vector comprising a polynucleotide that encodes a 3-ketosteroid reductase;

a plurality of host cells that are auxotrophic for cholesterol;

chemically defined, serum-free media;

growth supplements that support the growth of said plurality of host cells at low-seeding and clonal densities; and

at least one protocol to utilize said kit.

9. The kit of claim 8 , wherein said 3-ketosteroid reductase comprises a murine 3-ketosteroid reductase.

10. The kit of claim 8 , wherein said polynucleotide encoding said reductase comprises SEQ ID NO:1 or SEQ ID NO:2, or encodes an amino acid sequence comprising SEQ ID NO:3 or SEQ ID NO:4.

11. The kit of claim 8 , wherein said vector is a recombinant DNA expression vector.

12. The kit of claim 11 , wherein the recombinant DNA expression vector further comprises at least a first transcription unit for a product gene under control of the human cytomegalovirus promoter.

13. The kit of claim 8 , wherein said host cell is selected from the group consisting of NS-O, NS-I, and CHO-215.

14. The kit of claim 8 , wherein said host cell is an NS-O mouse myeloma cell.

15. The kit of claim 8 , wherein said host cells are adapted to chemically defined, serum-free medium.

16. The kit of claim 8 , wherein said host cells are adapted to chemically defined medium.

17. The kit of claim 8 , wherein said growth supplements comprise at least one of fatty acid-free BSA, rhIL-6, recombinant human insulin, sodium selenite, sodium pyruvate, and ethanolamine.

18. The kit of claim 8 , wherein said growth supplements comprise final concentrations in the chemically defined, serum free media of 0.1% to 5% fatty acid-free BSA, 1 ng/mL to 9 ng/mL rhIL-6, 5 mg/mL to 15 mg/L recombinant human insulin, 5 μg/L to about 8 μg/L sodium selenite, 0.01 g/L to 0.3 g/L sodium pyruvate, and 0.5 mg/L to 3.5 mg/L ethanolamine.

19. The kit of claim 8 , wherein said growth supplements comprise final concentrations in the chemically defined, serum free media of 1% fatty acid-free BSA, 5 ng/mL rhIL-6, 10 mg/L recombinant human insulin, 6.7 μg/L sodium selenite, 0.11 g/L sodium pyruvate, and 2.0 mg/L ethanolamine.

20. A composition of cell culture supplements comprising 0.1% to 5% fatty acid-free BSA, 1 ng/mL to 9 ng/mL rhIL-6, 5 mg/mL to 15 mg/L recombinant human insulin, 5 μg/L to 8 μg/L sodium selenite, 0.01 g/L to 0.3 g/L sodium pyruvate, and 0.5 mg/L to 3.5 mg/L ethanolamine.

21. The composition of claim 20 comprising 1% fatty acid-free BSA, 5 ng/mL rhIL-6, 10 mg/L recombinant human insulin, 6.7 μg/L sodium selenite, 0.11 g/L sodium pyruvate, and 2.0 mg/L mg/L ethanolamine.

22. A method of making a cell that is auxotrophic for cholesterol able to survive in cholesterol-free medium, the method comprising:

transfecting a eukaryotic cell that is auxotrophic for cholesterol with a vector comprising a polynucleotide that encodes a 3-ketosteroid reductase and optionally at least one polynucleotide that encodes a heterologous protein; and

wherein said polynucleotide that encodes the 3-ketosteroid is expressed by the transfected cell to confer the ability to survive in cholesterol-free medium.

23. The method of claim 22 , wherein said cells are selected from the group consisting of NS-0, NS-1, and CHO-215.

24. The method of claim 23 , wherein said cells are NS-O mouse myeloma cells.

25. The method of claim 22 , wherein said medium is chemically defined and serum-free or chemically defined.

26. A method for obtaining cells that have the ability to survive in a medium lacking cholesterol and have the ability to express a heterologous protein comprising:

transfecting eukaryotic cells that are auxotrophic for cholesterol with a vector comprising a polynucleotide encoding a 3-ketosteroid reductase, and at least one polynucleotide that encodes a heterologous protein; and

selecting the cells that have the ability to survive in medium lacking cholesterol.

27. The method of claim 26 , wherein said cells are selected from the group consisting of NS-O, NS-I, and CHO-215.

28. The method of claim 27 , wherein said cells are NS-O mouse myeloma cells.

29. The method of claim 26 , wherein said medium is chemically defined and serum-free or chemically defined.

30. A method of expressing a heterologous protein comprising transfecting a cell that is auxotrophic for cholesterol with a vector comprising a polynucleotide encoding a 3-ketosteroid reductase, wherein the vector further comprises a polynucleotide encoding the heterologous protein; and culturing the transfected cell in a cholesterol-free medium under conditions to provide expression of said heterologous protein.

31. The method of claim 30 , wherein said cells are selected from the group consisting of NS-O, NS-I, and CHO-215.

32. The method of claim 31 , wherein said cells are NS-O mouse myeloma cells.

33. The method of claim 30 , wherein said medium is chemically defined and serum-free or chemically defined.