Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Abstract

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/ MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

“…The FVII expression amount in the selected CHO-K1 cell line was determined to be approximately 300 ng/mL. These results suggest that the construction of vectors (Girod et al, 2005;Wang et al, 2008) should be used in future research to increase the copy number in CHO-K1 cells and thereby improve expression levels.…”

Cell-specific effects of human cytomegalovirus unique region on recombinant protein expression

“…Insulators are known to protect genes within a chromatin domain from stimulatory or repressive effects of flanking genomic regions (Antes et al 2001;Bode et al 2000;Namciu et al 1998). In addition, Girod et al (2005) showed that an insulating element, MAR, improved the transgene expression in stable producing cells. To determine the insulating activity of UR, we generated HEK293, HeLa, and CHO-K1 stable cells, which contain stably integrated CMV plasmids.…”

“…On the other hand, UR has been shown to act as an insulator (Angulo et al 2000;Lashmit et al 2004;Lundquist et al 1999). Insulating elements, such as nuclear matrix-associated regions (MARs), can enhance recombinant protein expression in stably integrated cell lines (Girod et al 2005;KohwiShigematsu et al 1997). Therefore, CMV UR could also be a positive element of CMV MIE promoter.…”

Cell line development for biomanufacturing processes: recent advances and an outlook

“…To minimize the influence of this position effect, cis-acting barrier elements, called insulators that block the propagation of a heterochromatin structure into neighboring euchromatin regions, have been incorporated into the expression vector to isolate the integrated transgene from nearby negative influences in the case where the vector is integrated into a condensed chromosomal region (Ghirlando et al 2012). The use of such insulating elements significantly increases the number of producing clones and improves the recombinant protein titer (Girod et al 2005;Boscolo et al 2012;Hou et al 2014).…”