Neomycin-phosphotransferase-genes and methods for the selection of recombinant cells producing high levels of a desired gene product
AssigneeBoehringer Ingelheim Pharma GmbH & Co. KG
InventorsBarbara ENENKEL, Juergen Fieder, Ralf Otto, Kerstin Sautter, Klaus Bergemann
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Abstract
The invention relates to modified neomycin phosphotransferase genes and their use in a selection method for high-producing recombinant cells. The invention further relates to expression vectors which contain a modified neomycin phosphotransferase gene and a gene of interest functionally linked to a heterologous promoter and a method of preparing heterologous gene products using these expression vectors.
Claims50
1-5. (canceled)
A modified neomycin phosphotransferase gene wherein the modified neomycin phosphotransferase gene compared with the wild-type gene encodes:
- glycine or aspartic acid at amino acid position 182 and/or alanine or valine or glycine at amino acid position 227 and/or glycine or asparagine at amino acid position 261 in relation to the wild-type gene.
(canceled)
(canceled)
A eukaryotic expression vector containing a heterologous gene of interest functionally linked to a heterologous promoter and a modified neomycin phosphotransferase gene which codes for a neomycin phosphotransferase having a lower enzyme activity compared with wild-type neomycin phosphotransferase.
A eukaryotic expression vector containing a heterologous gene of interest functionally linked to a heterologous promoter and a modified neomycin phosphotransferase gene which codes for a neomycin phosphotransferase having a lower enzyme activity compared with wild-type neomycin phosphotransferase wherein:
- (i) the modified neomycin phosphotransferase gene is a gene according to claim 1, or
- (ii) the modified neomycin phosphotransferase gene at amino acid position 182 and/or 227 codes for a different amino acid than the wild-type gene at the corresponding site, or
- (iii) the modified neomycin phosphotransferase gene at amino acid position 261 codes for a glycine.
(canceled)
(canceled)
A method of obtaining and selecting a mammalian cell which expresses at least one heterologous gene of interest, comprising:
- (i) transfecting a pool of mammalian cells with at least one gene of interest and a gene for a modified neomycin-phosphotransferase according to claim 1;
- (ii) cultivating the mammalian cells under conditions which allow expression of the gene of interest and expression of the modified neomycin-phosphotransferase gene;
- and
- (iii) cultivating the mammalian cells in the presence of at least one selecting agent which acts selectively on the growth of mammalian cells, and gives preference to the growth of the cells which express the modified neomycin-phosphotransferase gene.
The method according to claim 39, wherein that the mammalian cell is additionally transfected with a gene for an amplifiable selectable marker and the cell of step (iv) which is sorted by flow-cytometric analysis is subjected to at least one gene amplification step, wherein the amplifiable selectable marker gene is dihydrofolate-reductase (DHFR), and wherein the gene amplification is carried out by the addition of methotrexate.
A method of producing at least one protein of interest in a recombinant mammalian cell, comprising:
- (i) transfecting a pool of mammalian cells with at least one gene of interest and one gene for a modified neomycin-phosphotransferase according to claim 1;
- (ii) cultivating the cell under conditions which allow expression of the gene of interest and of the modified neomycin-phosphotransferase;
- (iii) cultivating the mammalian cell in the presence of at least one selecting agent which acts selectively on the growth of the mammalian cell, and gives preference to the growth of the cell which expresses the modified neomycin-phosphotransferase gene;
- and
- (iv) obtaining the protein of interest from the mammalian cells or the culture supernatant.
Dependencies are inferred from the claim text.