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Establishment of a stable CHO cell line with high level expression of recombinant porcine IFN-?.

ABSTRACT: A CHO cell clone (CHO-PoIFN-?) with stable porcine IFN-? expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-? reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-? could induce the expression of porcine Mx protein in PK15 cells, and activate the chicken Mx promoter-controlled luciferase reporter gene expression, confirming that the recombinant PoIFN-? has the biological activity of natural porcine type-I interferon. In addition, the recombinant PoIFN-? fully protected PK15 cells against 1000 TCID(50) of porcine transmissible gastroenteritis virus and pseudo-rabies virus infection, demonstrating its high potential in therapeutic applications. This is the first report of establishing a mammalian cell line with stable expression of porcine IFN-?.

SUBMITTER: Chen W

PROVIDER: S-EPMC7128424 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Establishment of a stable CHO cell line with high level expression of recombinant porcine IFN-β.

Chen Weiye W Cao Wenyan W Zhao Huijun H Hu Qianqian Q Qu Linmao L Hu Sen S Ge Jinying J Wen Zhiyuan Z Wang Xijun X Li Haobo H Huang Kehe K Bu Zhigao Z

Cytokine 20110401 3

A CHO cell clone (CHO-PoIFN-β) with stable porcine IFN-β expression under control of CMV promoter was selected under G418 pressure. In a 25cm(2) cell culture flask (5 ml culture medium), the cumulative protein yield of recombinant PoIFN-β reached 2.3×10(6) IU/ml. This cells clone maintained stable expression for at least 20 generations even in the absence of G418 selection pressure. The expressed recombinant PoIFN-β could induce the expression of porcine Mx protein in PK15 cells, and activate th ...[more]

PMID: 21459017

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Project description:Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise minimization may therefore be a novel strategy to reduce apparent expression instability and simplify cell line selection.

Project description:BackgroundChinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage.ResultsThe transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development.ConclusionsThe exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development.

Dataset Information

Establishment of a stable CHO cell line with high level expression of recombinant porcine IFN-?.

Publications

Establishment of a stable CHO cell line with high level expression of recombinant porcine IFN-β.

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