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.
Claims33
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.