NOVEL STRINGENT SELECTABLE MARKERS

摘要

The present invention relates to nucleic acid constructs comprising selectable marker genes in a multicistronic transcription unit for use in the generation and selection of eukaryotic host cells for expression of a gene product of interest. For increased stringency of selection, the coding sequence of the selectable marker may be directed preceded by a relatively short functional open reading frame to reduce the efficiency of translation of the selectable marker, and/or the amino acid sequence of the selectable marker may comprise one or more mutations that reduce the level of resistance provide by the mutated marker as compared to its wild type counterpart. The invention further relates to methods for generating eukaryotic host cells for expression of a gene product of interest, wherein these nucleic acid constructs are used, and to methods for producing a gene product of interest wherein thus generated host cells are applied.

主权项

1. A nucleic acid construct comprising a multicistronic transcription unit comprising:
a) a nucleotide sequence encoding a selectable marker functional in a eukaryotic host cell; b) a functional open reading frame comprising in a 5′ to 3′ direction a translation initiation codon, between 1 and 199 amino acid codons and a translation stop codon, and c) a nucleotide sequence coding for a gene product of interest upstream of the functional open reading frame, wherein the stop codon of the functional open reading frame is present between 0 and 300 nucleotides upstream of a translation initiation codon of the nucleotide sequence encoding the selectable marker, wherein the functional open reading frame encodes a non-functional peptide, and wherein the sequence separating the stop codon of the functional open reading frame and the separate translation initiation codon of the nucleotide sequence encoding the selectable marker is devoid of translation initiation codons.