INTRODUCTION TO GPEX TECHNOLOGY
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
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.