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Selection of host cells expressing protein at high levels

A technology of host cells and expression cassettes, applied in the field of molecular biology and biology, can solve the problems of unpredictable expression and inactivity of transgenes

Inactive Publication Date: 2009-03-18
CHROMAGENICS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] A problem with transgene expression is that it is unpredictable due to the high probability of the transgene becoming inactive due to gene silencing (McBurney et al., 2002), so in order to obtain high expression of the transgene, it is necessary to test numerous host cell cloning

Method used

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  • Selection of host cells expressing protein at high levels
  • Selection of host cells expressing protein at high levels
  • Selection of host cells expressing protein at high levels

Examples

Experimental program
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Effect test

Embodiment 1

[0090]Example 1 describes a selection system with a polycistronic transcription unit of the invention, and it will be apparent that variations described in Examples 8-26 of WO 2006 / 048459 incorporated herein by reference can also be used and tested for the polycistronic transcription unit of the invention. Cistronic transcription unit. The same is true for Examples 20-27 of US 2006 / 0195935.

[0091] Example 1: Stringent selection by placing a modified Zeocin resistance gene behind an IRES sequence Examples 8-26 of WO 2006 / 048459 (incorporated herein by reference in its entirety) have shown a selection system in which multiple The sequence encoding the selectable marker protein on the cistron transcription unit is upstream of the sequence encoding the protein of interest, where the translation initiation sequence of the selectable marker is non-optimal, and where the remaining internal ATG has been removed from the selectable marker coding sequence. This system results in a hi...

Embodiment 2

[0098] Example 2: Stability of expression after placing the modified dhfr gene in the IRES sequence

[0099] The translation initiation codon of the Zeocin selectable marker was modified to a translation initiation codon that is used much less frequently than the usual ATG codon, resulting in a high stringency selection system. In the selection system described in WO2006 / 048459, TTG Zeo is placed upstream of the gene of interest. In another possible selection system, a Zeo selection marker is placed downstream of the IRES sequence (this application, see Example 1). This produces a bicistronic mRNA from which the Zeo gene product is translated from the translation initiation codon in the IRES sequence.

[0100] In this experiment, we combined implementations of these two systems. We placed a TTG selection marker upstream of the reporter gene and coupled a GTG or TTG-modified metabolic marker with an IRES to the reporter gene. Different selectable marker genes can be used, su...

Embodiment 3

[0117] Example 3: The increased expression of the modified dhfr gene following placement of a weakened IRES sequence is not a result of gene amplification.

[0118] In the prior art, the use of the dhfr gene as a selection marker usually relies on the amplification of the dhfr gene. A toxic agent, methotrexate, is used in this system to amplify the dhfr gene, along with the desired transgene, of which up to several thousand copies can be integrated into the genome of CHO cells following such amplification . Although these high copy numbers yielded high expression levels, they were also considered a disadvantage because so many copies could cause increased genome instability and the subsequent removal of methotrexate from the medium would cause many amplifications to occur. Rapid removal of augmented loci.

[0119] In Example 2, no methotrexate was used to inhibit dhfr enzyme activity. Only hypoxanthine and thymidine precursors are removed from the medium, which is sufficien...

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Abstract

The invention provides a DNA molecule comprising a multicistronic transcription unit coding for i) a polypeptide of interest, and for ii) a selectable marker polypeptide functional in a eukaryotic host cell, wherein the polypeptide of interest has a translation initiation sequence separate from that of the selectable marker polypeptide, and wherein the coding sequence for the polypeptide of interest is upstream from the coding sequence for the selectable marker polypeptide in the multicistronic transcription unit, and wherein an internal ribosome entry site is present downstream from the coding sequence for the polypeptide of interest and upstream from the coding sequence for the selectable marker polypeptide, and wherein the nucleic acid sequence coding for the selectable marker polypeptide in the coding strand comprises a GTG or a TTG start codon. Also provided are methods for obtaining host cells expressing a polypeptide of interest, such host cells comprising DNA molecules of the invention. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules of the invention.

Description

field of invention [0001] The present invention relates to the fields of molecular biology and biotechnology. More specifically, the present invention relates to methods and means for improving the selection of host cells expressing proteins at high levels. Background of the invention [0002] Proteins can be produced in various host cells and are widely used in biology and biotechnology, such as biopharmaceuticals. It is preferred that eukaryotic, especially mammalian, host cells express proteins, for example when such proteins have certain post-translational modifications such as glycosylation. Methods for such production are well established and generally entail expression in a host cell of a nucleic acid encoding the protein of interest (also referred to as a "transgene"). Typically, the transgene is introduced into precursor cells along with a selectable marker gene, cells are selected based on expression of the selectable marker gene, and one or more clones expressin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/65C12N15/85C12N2830/40C12N2840/00C12N2840/203C12P21/02C12N15/11C12N15/63C12N15/09
Inventor A·P·奥特H·J·M·范布洛克兰T·H·J·克瓦克斯R·G·A·B·西沃尔特
Owner CHROMAGENICS BV
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