Compositions and Methods for Metabolic Selection of Transfected Cells
AssigneeBiofactura, Inc.
InventorsLuis Branco, Darryl Sampey
Life of this patent
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Abstract
The present invention relates to novel selection marker vectors, and methods for using these vectors to generate stable gene expression systems in eukaryotic cells utilizing any enzyme useful in the eukaryotic sterol/cholesterol biosynthetic pathway, such as a 3-ketosteroid reductase, as a metabolic selection marker to select transfected cells. In one embodiment, the method comprises transfecting cells that are auxotrophic for cholesterol with a vector encoding 3-ketosteroid reductase and at least one heterologous protein, and selecting cells that have the ability to survive in medium lacking cholesterol and/or producing the heterologous protein in these cells in chemically defined and/or serum-free media.
Claims43
A vector comprising a polynucleotide encoding a 3-ketosteroid reductase and a polynucleotide encoding a heterologous polypeptide.
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A kit comprising:
- a vector comprising a polynucleotide that encodes a 3-ketosteroid reductase;
- and optionally one or more of:
- 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.
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A composition of cell culture supplements comprising about 0.1% to about 5% fatty acid-free BSA, about 1 ng/mL to about 9 ng/mL rhIL-6, about 5 mg/mL to about 15 mg/L recombinant human insulin, about 5 μg/L to about 8 μg/L sodium selenite, about 0.01 g/L to about 0.3 g/L sodium pyruvate, and about 0.5 mg/L to about 3.5 mg/L ethanolamine.
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 reductase is expressed by the transfected cell to confer the ability to survive in cholesterol-free medium.
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Dependencies are inferred from the claim text.