The first step in the process of generating a high expressing mammalian cell line for production of therapeutic recombinant protein is developing a robust expression vector that is compatible with the host cell line of choice. Transcription of the recombinant gene will largely depend on the strength of the expression vector and the site of genomic integration of the vector. The promoter, enhancer, poly-A sequence, and the stringency of the selection marker will all contribute to the overall strength of the expression vector, although the degree of contribution from each component may vary. In addition, use of various genomic DNA elements such as Expression Augmenting Sequence Element (EASE), scaffold- or matrix-attachment regions (S/MAR elements), Insulators, or Universal Chromatin Opening Element (UCOE), can further improve recombinant protein expression by protecting transgenes from inactivation by flanking chromatin sequences. The use of chromatin remodeling genomic DNA elements can also reduce variations in the expression levels of the same construct in individual clones, thus facilitating often labor-intensive clone screening process. Similar to promoters and enhancers, chromatin remodeling elements contain binding sites for transcription factors, however, these transcription factors tend to have relaxed sequence requirements or low-affinity binding characteristics individually, which can result in stable DNA interactions through cooperative binding with other transcriptional factor partners (Zuckerkandl and Villet, 1988).
In addition to the choice of promoter, enhancer, or genomic DNA elements used in the vector, the order, orientation, and distances between the individual components can have a profound effect on the final activity of the vector, and subsequently expression of the recombinant protein. This is not surprising since cooperative effects of relatively low-affinity DNA binding factors will depend on proper position or distribution of each binding site, compatible with precisely positioned high order structure, required for the transcription initiation complex.