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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
  • C12N15/69—Increasing the copy number of the vector

Definitions

• Figure 8 shows the correlation between antibody productivity (mAb F19) and GFP fluorescence using the cell pool ZB1 as an example.
• This cell pool was obtained from transfection with the vector combination pBIDG-F19HC and pBIN-F19LC (Fig.3).
• the pool was subjected to a sequential GFP-based FACS sorting. After each sort, the concentration of the antibody F19 in the cell culture supernatant of the pool was determined by ELISA and the specific productivity per cell and day (pg / c * d) was calculated. Each data point represents the average of at least three cultivation passages. A total of six sorts were carried out.
• Modified promoters which have a minimal sequence homology to the wild-type sequence SEQ ID NO: 1 of the hamster ubiquitin / S27a promoter of at least 80%, better at least 85%, preferably at least 90%, more preferably at least 95% and particularly preferred are particularly preferred have at least 97% and have a corresponding promoter activity in a comparative reporter gene assay.
• nucleotide sequence or “nucleic acid sequence” denotes an oligonucleotide, nucleotides, polynucleotides and their fragments as well as DNA or RNA of genomic or synthetic origin, which are present as a single or double strand and can represent the coding or the non-coding strand of a gene.
• Standard techniques such as, for example, site-specific mutagenesis or PCR-mediated mutagenesis (for example described in Sambrook et al., 1989 or Ausubel et al., 1994) can be used to modify nucleic acid sequences.
• a person skilled in the art refers to a bivalent homodimeric scFv derivative as “diabody”.
• the shortening of the peptide linker in the scFv molecule to 5-10 amino acids results in the formation of homodimers through the superposition of VH / VL chains.
• the diabodies can also be stabilized by the introduction of disulfide bridges. Examples of diabodies can be found in the literature, for example in Perisic et al. (1994).
• fragments which the person skilled in the art calls mini-antibodies and which have a bi-, tri- or tetravalent structure are also derivatives of scFv fragments.
• the multimerization is achieved via di-, tri- or tetrameric “coiled coil” structures (Pack et al., 1993 and 1995; Lovejoy et al., 1993).
• the fluorescence proteins used according to the invention also include natural or genetically engineered mutants and variants, their fragments, derivatives or e.g. Variants fused with other proteins or peptides.
• the mutations introduced can change, for example, the excitation or emission spectrum, the formation of chromophores, the extinction coefficient or the stability of the protein. Codon optimization can also improve expression in mammalian cells or other species.
• the fluorescent protein can also be used in fusion with a selection marker, preferably with an amplifiable selection marker such as, for example, dihydrofolate reductase (DHFR).
• DHFR dihydrofolate reductase
• enhancers are known to the person skilled in the art from various sources (and are stored in databases such as GenBank, for example SV40 enhancer, CMV enhancer, polyoma enhancer, adenovirus enhancer) and are available as independent elements or elements cloned within polynucleotide sequences (e.g. deposited at ATCC or from commercial and individual sources).
• a variety of promoter sequences also include enhancer sequences, e.g. the commonly used CMV promoter.
• the human CMV enhancer is one of the strongest enhancers identified to date.
• An example of an inducible enhancer is the metallothionein enhancer, which can be stimulated by glucocorticoids or heavy metals.
• inducible promoters the activity of the promoter can be reduced or enhanced in response to a signal.
• An example of an inducible pro motor is the tetracycline (tet) promoter.
• tet tetracycline
• tetO tetracycline operator sequences
• tTA tetracycline-regulated transactivator protein
• jun, fos, metallothionine and heat shock promoters see also Sambrook et al., 1989; Gossen et al., 1994).
• Table 1 below gives examples of further amplifiable selection marker genes which can be used according to the invention and the associated selection agents, which are described in an overview by Kaufman, Methods in Enzymology, 185: 537-566 (1990).
• the concentration of MTX in the first amplification step is preferably at least 200 nM, in an even more preferred embodiment at least 500 nM and can be increased in steps up to 1 / M. In individual cases, concentrations of over 1 ⁇ M can also be used.
• PCR polymerase chain reaction sICAM: soluble intracellular adhesion molecule
• the therapeutic protein sICAM and GFP were expressed together by a bicistronic and the DHFR by a separate transcription unit. Two to three weeks after the first selection in HT-free CHO-S-SFMII medium, the 5% of the cells with the highest GFP fluorescence were sorted out. After approximately two weeks of cultivation, the 5% cells with the highest GFP fluorescence were again isolated. In total, this sequential sorting was carried out six times. A good correlation between sICAM productivity and GFP fluorescence was shown (Fig. 4). The FACS-based selection alone, without any MTX amplification step, quickly isolated cell pools with high specific productivities of up to 16 pg / cell / day (Fig. 5).

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