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Synthetic promoters for CHO cells, and methods of producing synthetic promoters using transcription factor binding site modules

A technology of transcription factor regulation and promoter, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as limited and discontinuous expression levels

Inactive Publication Date: 2016-07-20
UCB PHARMA SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While this is a promising approach to identifying promoters with discrete expression levels, it is limited to naturally occurring activities
Furthermore, defining the genomic regulatory sequences that control the expression of specific genes can be a significant challenge

Method used

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  • Synthetic promoters for CHO cells, and methods of producing synthetic promoters using transcription factor binding site modules
  • Synthetic promoters for CHO cells, and methods of producing synthetic promoters using transcription factor binding site modules
  • Synthetic promoters for CHO cells, and methods of producing synthetic promoters using transcription factor binding site modules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0328] Example 1: Identification of active transcription factor regulatory elements in CHO-S cells

[0329] In silico analysis of transcription factor regulatory elements

[0330] To identify discrete TFREs (transcription factor binding sites) capable of transactivating recombinant genes in CHO-S cells, the inventors surveyed putative TFREs in 10 viral promoters generally known to be active in CHO cells .

[0331] The following promoter sequences were retrieved from the GenBank database: hCMV-IE1 (accession number M60321.1), mouse CMV-IE1 (M11788), rat CMV-IE1 (U62396), guinea pig CMV-IE1 (CS419275), small Murine CMV-IE2 (L06816.1), Simian virus 40 early promoter and enhancer (NC_001669.1), Adenovirus major late promoter (KF268310), Myeloproliferative sarcoma virus long terminal repeat (LTR) (K01683.1) , Rous sarcoma virus LTR (J02025.1) and human immunodeficiency virus LTR (K03455.1).

[0332] Using the Transcriptional Element Search System (TESS: http: / / www.cbil.upenn...

Embodiment 2

[0348] Example 2: Construction and analysis of the 1st generation promoter

[0349] Synthetic promoter library construction

[0350] Synthetic promoter TFRE was constructed from complementary single-stranded 5' phosphorylated oligonucleotides (Sigma, Poole, UK) by heating at 95 °C for 5 minutes, followed by slow cooling to 25 °C over 2 hours, in STE buffer (100 mM NaCl , 50mM Tris-HCl, 1mM EDTA, pH7.8, Sigma) annealed. Oligonucleotides were designed such that the resulting double-stranded blocks contained specific TFREs (Table 1), and 4 bp TCGA single-stranded overhangs at each 5' end. For example, the sequence for the NFkB-RE block is as follows (RE site underlined): 5'-TCGAT GGGACTTTCC A - 3' SEQ ID NO: 171 and 5'-TCGAT GGAAAGTCCC A-3' SEQ ID NO: 172.

[0351] For construction of the first generation synthetic promoter library, all seven TFREs identified as transcriptionally active in CHO-S cells were used. Oligonucleotide building blocks containing a single copy ...

Embodiment 3

[0367] Example 3: Construction and analysis of the second generation synthetic promoter

[0368] Based on the above analysis of the 1st generation synthetic promoters, the present inventors sought to further improve Synthetic promoter activity.

[0369] Using the initial seven identified TFREs (see figure 1 ) were used to construct a library of 2nd generation synthetic promoters with a stoichiometry derived quantitatively from their relative representation among the active synthetic promoters in the 1st generation synthetic promoter constructs. The stoichiometric ratio used was 5:3:1:1 (NFkB-RE:E-box:C / EBPa-RE:GC-box).

[0370] Specifically, of the original seven TFREs, negative TFREs, E4F1 and CRE (see Figure 3) had been omitted (i.e. promoters containing greater numbers of these TFREs were associated with lower expression levels of reporter genes), Whereas the neutral TFRE, C / EBPa and GC-boxes were included on the assumption that increased complexity might be advantageo...

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Abstract

CHO cell-specific synthetic promoter constructs for expressing recombinant proteins, a library of promoter constructs thereof, and a method for producing the promoter constructs. The promoter constructs enable precise control of recombinant gene transcription over three orders of magnitude, with the top expressing promoters capable of double the transcriptional activity of the CMV promoter.

Description

[0001] The present invention relates to synthetic promoter constructs for use in mammalian cells, particularly Chinese Hamster Ovary (CHO) cells, and recombinant host cells comprising the synthetic promoter constructs. The invention further relates to methods for the production of these promoter constructs, said recombinant cells, and the use of any of these synthetic promoter constructs, said recombinant cells, for example in the expression of recombinant proteins. Background technique [0002] Chinese hamster ovary (CHO) cells are derived from the ovaries of Chinese hamsters and are widely used in genetic research, toxicity screening and gene expression, especially for the expression of recombinant proteins. They were first introduced in the 1960s, grow in suspension culture, and require proline in their medium. [0003] CHO cells are the most commonly used mammalian host cell line for large-scale production of recombinant protein therapeutics. Therefore, CHO cells are impo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/10C12N15/113C12N15/85C40B50/06
CPCC12N5/00C12N15/85C40B50/06C12N2310/10C12N2800/40C12N2800/24C12N2830/00C12N2830/15C12N2830/008C12N15/1051C12N15/1086C12N15/1058
Inventor D·C·詹姆斯A·J·布朗
Owner UCB PHARMA SA
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