Development of stable cell lines for production or regulated expression using matrix attachment regions
Introduction
Selection and screening procedures to identify the prized clone with the requisite expression characteristics for regulated expression or production are tedious and time-consuming. In Chinese hamster ovary (CHO) cells, the classical approach to achieve maximal expression involves the use of mutant cell lines and a gradual increase in the selection pressure over several months for a co-transfected selection marker such as dihydrofolate reductase (Kaufman and Sharp, 1982, Schimke et al., 1982). While new approaches to the problem include the identification of rare sites on the chromosome with high transcriptional activity combined with targeted integration, the improvement of selection and of screening procedures (Fussenegger et al., 1999), these are all labor-intensive.
The variability in expression levels is thought to reflect the influence of the chromatin structure and/or the presence of regulatory elements at the site of integration in the host genome, a phenomenon referred to as ‘position effect’. A simple and rapid approach to overcome position effects would be to make use of chromatin elements that prevent the neighboring chromatin from affecting transgene expression. This is expected to improve the chances of isolating a clone exhibiting the desired regulated expression for ex vivo gene therapy, or high-level expression for production of a recombinant protein. Consequently, the time spent screening clones would be considerably reduced. Furthermore, the position-independent transgene expression has significant potential in the construction of regulated gene expression systems, as the expression of a therapeutic gene and its controlling components would be independent of the chromatin structure at the integration site. Chromatin elements potentially capable of overcoming position effects, and hence of interest for the development of stable cell lines, include boundary elements (BEs), scaffold or matrix attachment regions (S/MARs), and locus control regions (LCRs).
BEs or insulator elements in many cases define boundaries in chromatin (Bell and Felsenfeld, 1999, Udvardy, 1999) and may have the role of defining a transcriptional domain in vivo. They lack intrinsic promoter/enhancer activity, but rather are thought to protect genes from the transcriptional influence of regulatory elements in the surrounding chromatin. The enhancer-block assay is commonly used to identify insulator elements. In this assay, the element is placed between an enhancer and a promoter, and enhancer-activated transcription is measured. BEs have been shown to be able to protect stably transfected reporter genes against position effects in Drosophila, yeast and in mammalian cells (Cuvier et al., 1998, Bi and Broach, 1999, Walters et al., 1999) and to increase the proportion of transgenic mice with inducible transgene expression (Wang et al., 1997).
S/MARs are DNA sequences that bind isolated nuclear scaffolds or nuclear matrices in vitro with high affinity (Hart and Laemmli, 1998). As such, they may define boundaries of independent chromatin domains, such that only the encompassing cis-regulatory elements control the expression of the genes within the domain. However, their ability to fully shield a chromosomal locus from nearby elements, and thus confer position-independent gene expression, was not seen in stably transfected cells (Poljak et al., 1994). On the other hand, S/MAR sequences have been shown to interact with enhancers to increase local chromatin accessibility (Jenuwein et al., 1997). S/MAR elements can enhance expression of heterologous genes in cell culture lines (Phi-Van et al., 1990, Klehr et al., 1991, Poljak et al., 1994, Kalos and Fournier, 1995), transgenic mice (Castilla et al., 1998) and plants (Allen et al., 1996). The utility of S/MAR sequences for developing improved vectors for gene therapy is starting to be recognized (Agarwal et al., 1998).
LCRs are cis-regulatory elements required for the initial chromatin activation of a locus and subsequent gene transcription in their native locations (reviewed in Grosveld, 1999). The activating function of LCRs also allows the expression of a coupled transgene in the appropriate tissue in transgenic mice, irrespective of the site of integration in the host genome. While LCRs generally confer tissue-specific levels of expression on linked genes, efficient expression in nearly all tissues in transgenic mice has been reported for a truncated human T-cell receptor LCR (Ortiz et al., 1997) and the rat LAP LCR (Talbot et al., 1994). The most extensively characterised LCR is that of the globin locus, and its use in vectors for the gene therapy of sickle-cell disease and β-thalassemias is currently being evaluated (Pawliuk et al., 1998).
In this study, single chromatin elements, as well as combinations of elements, were tested for their capacity to increase stable transgene expression in industrially relevant CHO cells. The chicken lysozyme 5′ MAR was the only element to significantly enhance reporter expression in pools of stable clones. We have thus evaluated the application of this MAR to generate stable cell lines for the production of recombinant proteins and for regulated transgene expression for encapsulated cell based therapies.
Section snippets
Plasmids construction
The luciferase expression vectors used to test the chromatin elements are all based on pGL3-Control (Promega). This plasmid contains an SV40 promoter in front of a modified firefly luciferase cDNA, followed by the SV40 late poly(A) signal and the SV40 enhancer. The Drosophila melanogaster elements come from the p7, p83 and p1314 plasmids kindly provided by Ulrich Laemmli (Poljak et al., 1994). The 1.8 kb SalI scs (special chromatin structure) BE fragment comes from p83, as well as the 960 bp Bam
Chromatin elements and stable transgene expression in CHO cells
The use of structural chromatin components to overcome silencing of stably integrated genes by the chromosomal environment would be particularly useful in biotechnology. Unfortunately, fully characterized chromatin elements in higher eukaryotes are rare, and most of these have not been tested with a heterologous promoter in heterologous cells. Elements which counteract the effect of neighboring chromatin structure on stable transgene expression are expected to raise the average transgene
Discussion
To date, the development of stable cell lines has been hampered by the negative effects of surrounding chromatin on the expression of randomly integrated vector sequences. Chromatin elements, such as BEs, MARs, and LCRs, are known to exert an effect on gene expression only when integrated in the genome. While the use of chromatin elements in the next generation of gene therapy vectors is currently being considered to improve expression of therapeutic transgenes (Neff et al., 1997), few studies
Acknowledgements
We thank Ulrich Laemmli for gift of the Drosophila elements, Wolf Strätling for the chicken lysozyme MAR, Ueli Schibler for the rat LAP LCR, and Astar Winoto for the human TCRα LCR. We are grateful to Hanspeter Amstutz for critical reading of the manuscript. This work was supported by the Swiss National Fund Swiss Priority Program in Biotechnology and National Research Program on Somatic Gene Therapy, and by the Etat de Vaud.
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