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Rodent expression systems utilising polyoma virus and epstein barr virus sequences

A technique for rodents, expression systems, applied in the field of protein expression systems

Inactive Publication Date: 2007-02-07
艾塞特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, previously described episomal vectors, such as those based on the Epstein-Barr virus (EBV) or SV40 large T antigen, have proven to be limited in host cell range and maintenance in long-term culture (Piechaczek et al. (1999); and Heffernan and Dennis (1991))

Method used

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  • Rodent expression systems utilising polyoma virus and epstein barr virus sequences
  • Rodent expression systems utilising polyoma virus and epstein barr virus sequences
  • Rodent expression systems utilising polyoma virus and epstein barr virus sequences

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Experimental program
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Effect test

Embodiment 1

[0087] Materials and methods:

[0088] cells and media

[0089] CHO-K1 (ATCCCCL 61) was grown in DMEM:COONS F12 containing 10% FBS. CHO-T cells were adapted for suspension growth in serum-free medium (Excell302; JRH Biosciences, Kansas) and protein-free medium (Excell325; JRH Biosciences, Kansas). XL99 cells (a suspension CHO-K1 cell line suitable for suspension growth in serum free medium (Neil Kitchen 1999)) were grown in Excell302.

[0090] NS0-T

[0091] Plasmids encoding the polyoma virus large T antigen were transfected with NSO cells (non-secreting mouse myeloma cells) (ECACC 85110503).

[0092] Electroporation of NS0

[0093] Electroporation of NSO cells was performed using the appropriate protocol described by Chu G et al. 1987. In short, put 1-1.5 x 10 7 NS0 cells were grown to mid-log phase and electroporated with 10 μg pBK-CMV-large T antigen plasmid at 250V with 15 ms square wave pulses. Cells were selected in G418 (400 μg / ml) 48 hours after transfection. ...

Embodiment 2

[0124] Episomal replication in CHO cells

[0125] The expression vector pPyOriLT ( Figure 1G ) into CHO cells to demonstrate episomal replication. Thus, pPyOriLT contains PyOri and encodes PyLT, two essential viral elements, and is sufficient to initiate plasmid DNA replication in the presence of permissive CHO cytokines. Monitor plasmid DNA replication for 3 days. Purify low-molecular-weight DNA from transfected cells according to the Hirt extraction technique described above ( Image 6 (1)). Plasmid replication is detected by resistance to DpnI, which cleaves only when its recognition site is methylated. DNA purified from E. coli dam+ strains (lanes 1 to 4) is a substrate for DpnI (lane 5), whereas plasmid DNA undergoing one or more rounds of DNA replication in mammalian cells is resistant to DpnI cleavage (lanes 11-20) . as in Image 6 As shown in (2), the total amount of non-replicating DNA (Dpnl sensitive) dropped rapidly from 7000 copies per cell to less than 100...

Embodiment 3

[0127] (a) Generation of CHO-T cell lines

[0128]The cDNA for the Py large T antigen was obtained from ATCC and cloned into the expression vector pBK-CMV (Clonetech). CHO-K1 (ATCC, CCL61) was transfected with the expression plasmid pBK-CMV-LT (Figure 1). Cells were selected in G418 (400 μg / ml) for two weeks. by as in figure 2 The immunofluorescence staining shown in further confirmed the expression of Py large T antigen. Single cells were isolated by fluorescence-activated cell sorting using a MoFlo cytometer (Dako-Cytomation, Fort Collins, Colorado, USA). separated as in image 3 Several clones with varying levels of large T antigens are shown. 21 clones with different large T antigen expression levels were expanded and Figure 4 Their ability to support DNA replication of a Py ori-containing plasmid (pNK-Ori-EGFP) was tested as shown in the Southern blot. pNK-Ori-EGFP was transfected into CHO-T clones. Low molecular weight DNA was extracted by the Hirt extraction me...

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Abstract

The present invention relates to protein expression systems and in particular to rodent cell expression systems utilising Polyoma Virus and Epstein Barr Virus elements. The invention utilises the large T antigen and origin of replication of polyoma virus and the EBV nuclear antigen-1 (EBNA-1) and EBV origin of replication from EBV. The present invention provides a rodent cell line with enhanced protein production capabilities. The invention also relates to eukaryotic cloning and expression vectors and related methods, and in particular to DNA vectors capable of high level expression of a protein of interest. The invention allows for long-term episomal maintenance of expression vectors in mammalian cells. The invention also relates to a method for increasing resistance to apoptosis in a rodent cell comprising expression of a polyoma large T antigen.

Description

technical field [0001] The present invention relates to protein expression systems, particularly mammalian protein expression systems. Specifically, the present invention provides rodent cell lines with enhanced protein production capacity. [0002] The present invention also relates to eukaryotic cloning and expression vectors and related methods, in particular to DNA vectors capable of expressing proteins of interest at high levels. The present invention allows expression vectors to remain episomal in mammalian cells for long periods of time. Background technique [0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known in the art or part of the common general knowledge. [0004] In recombinant protein production, the choice of host cells is very important. Expression systems for many recombinant proteins are available, including bacterial, yeast, fungal, insect, plant and mam...

Claims

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Application Information

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IPC IPC(8): C12N15/85
Inventor N－A·松斯特伦R·库纳普拉朱
Owner 艾塞特生物技术有限公司
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