Asparaginase based selection system for heterologous protein expression in mammalian cells
AssigneesThe Regents of the University of California, Technical University of Denmark
InventorsHooman Hefzi, Nathan E. Lewis, Karen Julie la Cour Karottki, Helene Faustrup Kildegaard
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Abstract
A non-naturally occurring cell comprising an inoperative genomic asparaginase (Aspg) gene and an inoperative glutamine synthetase (Gs) gene, wherein the cell has been transfected with a controllably expressed gene encoding an enzyme having asparaginase activity, a controllably expressed gene encoding an enzyme having glutamine synthetase activity, and a controllably expressed gene encoding a heterologous protein of interest.
Claims20
A non-naturally occurring mammalian cell comprising an inoperative genomic asparaginase (Aspg) gene and an inoperative glutamine synthetase (Gs) gene, wherein the cell has been transfected with a controllably expressed gene encoding an enzyme with asparaginase activity, a controllably expressed gene encoding an enzyme with glutamine synthetase activity, and a controllably expressed gene encoding a heterologous protein.
A selection system for heterologous protein expression in cells, comprising a non-naturally occurring mammalian cell comprising an inoperative genomic asparaginase (Aspg) gene and an inoperative glutamine synthetase (Gs) gene, wherein the cell has been transfected with a controllably expressed gene encoding an enzyme with asparaginase activity, a controllably expressed gene encoding an enzyme with glutamine synthetase activity, and a controllably expressed gene encoding a heterologous protein;
- and
- a medium which contains substantially no glutamine.
A method of preparing a non-naturally occurring mammalian cell for production of a heterologous protein, comprising:
- a) contacting a mammalian cell with a gene editing system configured to inactivate an asparaginase (Aspg) gene and a glutamine synthetase (Gs) gene;
- and
- b) transfecting the mammalian cell or a descendent of the mammalian cell with:
- i) a first plasmid comprising a controllably expressed gene encoding an enzyme with asparaginase activity and a first controllably expressed gene encoding the heterologous protein;
- and
- ii) a second plasmid comprising a controllably expressed gene encoding an enzyme with glutamine synthetase activity and a second controllably expressed gene encoding the heterologous protein.
Dependencies are inferred from the claim text.