Consistent Production of Genetically Stable Mammalian Cell Lines - The author describes expression technology that produces cell lines with high genetic stability. This article is part of a special se

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Consistent Production of Genetically Stable Mammalian Cell Lines
The author describes expression technology that produces cell lines with high genetic stability. This article is part of a special section on expression systems.


BioPharm International
Volume 25, Issue 5, pp. 56-59

INTRODUCTION TO GPEX TECHNOLOGY


Figure 1
GPEx is a unique and versatile system designed to insert genes of interest into a wide variety of mammalian host cells. The GPEx method is based on the use of replication defective retroviral vectors (retrovectors) to actively insert the desired genes into the genome of dividing cells. The majority of the components from GPEx retrovectors are derived from Moloney Murine Leukemia virus (MLV). The Vesicular Stomatitis Virus G Protein (VSV-G) is used as an envelope for the retrovector particle. These particles stably insert single copies of the transgene at multiple sites in the chromatin of dividing cells. Retrovectors deliver genes coded as RNA that, after entering the cell, are reverse transcribed to DNA and integrated stably into the genome of the host cell (see Figure 1). Two enzymes, reverse transcriptase and integrase, provided transiently in the vector particle, perform this function. These integrated genes are maintained through subsequent cell divisions as if they were endogenous cellular genes. By controlling the number of retrovector particles accessing the cell, multiple gene insertion can be achieved without traditional amplification steps.

HIGH EXPRESSION AND GENETIC STABILITY PROPERTIES

Each transgene copy is inserted at a unique site in the chromosome

GPEx gene insertions occur at unique locations in the cell genome, with a single copy of the gene being inserted at each independent site. Unlike most other methods of transgene insertion that are undefined "passive" processes, each insertion by a retrovector is an "active" process that is modulated by the MLV integrase enzyme (5). This method prevents head-to-tail arrays at a single site in the host cell genome.

The technology inserts genes into a wide variety of cell lines

The method uses VSV-G as an envelope on the retrovector particles. This envelope protein allows the retrovectors to insert genes into all mammalian cells, in addition to numerous other cell types, because of its ability to bind to various membrane phospholipids and glycolipids (6–8).

Transgene inserts target open or active regions of the cell genome

The MLV based retrovectors have been shown to preferentially insert into or around the transcription start point of genes (9, 10). This preference for transcriptionally active regions of the genome allows for higher, more consistent levels of expression per copy of the gene inserted compared to other methods of gene insertion.

Amplification without antibiotic selection or use of toxic compounds


Figure 2
Because of the extremely high gene insertion efficiency of the GPEx process, no selectable markers (e.g. neomycin, blasticidin, hygromycin, or puromycin resistance genes) are needed for cell line generation. This has a number of advantages over other cell line development methods, including reduced costs for culturing cells, no additional taxing of the cells, and reduced time for clonal cell line selection. The high transduction efficiency and the ability to do repeat cell transductions generate high copy number cell lines, thereby eliminating the need to amplify gene copy number by adding toxic compounds, such as methotrexate or methionine sulphoximine. To increase copy number, retransduction of a cell line is performed yielding clonal lines with copy numbers ranging from 25–250.


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