Generation of a novel host cell line by combining the strengths of recombinase−mediated cassette exchange with BAC expression vectors

Background.

Elucidation of bottlenecks in recombinant protein expression is impeded amongst others by biological limitations due to differences in the transgene integration site or adaptive responses and gene translocation during drug selection. To circumvent these problems we have generated our own host cell line carrying two heterospecific, non−compatible flippase (Flp) recognition target sites (FRT) (Turan et al., 2013). Compared to commercially available recombination−competent host cell lines our newly developed cell line contains two heterospecific recombinase recognition sites providing higher specificity during transgene integration. This system was already tested with two different proteins and the generated stable clones were evaluated positively for homologous recombination and product secretion (Mayrhofer et al., submitted).

Designing appropriate expression vectors is one of the critical steps in the generation of stable cell lines for recombinant protein production. The question as to whether a transgenic integration site may be transcriptionally active or not will ultimately determine the overall productivity of a newly generated cell line. To bypass this so called “position effect” we applied a 200-kbp Bacterial Artificial Chromosome (BAC) construct containing the eukaryotic chromosomallocus Rosa26 (Thumpd3)(Zambrowicz et al., 1997; Blaas et al., 2009). The Rosa26 locus is a region known to consist of open chromatin and is widely used for gene expression in transgenic mice. By placing an expression cassette (promoter, gene of interest, polyadenylation signal and selection marker) into the Rosa26 BAC it was possible to significantly increase the productivity in stably transfected HEK293 and CHO cells as compared to the conventionally used plasmid-based vectors that are prone to “position effects” (Kunert and Casanova, 2013; Mader et al., 2013).

Aims and methods.

The aim of this project is to generate new host cell lines, which are capable of integrating transgenes via homologous recombination into a defined locus. This defined locus will be the Rosa26 mouse open chromatin element, which is integrated on a BAC vector. Further, we will insert flippase recognition sequences flanking the expression cassette to generate a promoter trap. The promoter trap is superior to other systems as it allows only correctly recombined, but not randomly integrated vectors, to be transcribed. The established host cell lines will be screened for growth properties and maximum achievable cell densities. Next recombinant cell clones will be generated and evaluated for their expression potential of different single chain antibodies. Afterwards we will also generate host cell lines for multiple gene integration events as needed for antibody expression.

The tool of homologous recombination in a homogeneous host cell line will enable elimination of biological limitations due to differences in the transgene integration site or adaptive responses during drug selection. By using this approach the usual formation of a heterogeneous pool of randomly integrated genes into different chromosomal loci can be reduced significantly. The new host cell lines will allow a more appropriate comparison of different transgenes independent of the integration locus. We will perform transcriptomic and proteomic analyses from different clones of individual transfections to screen for molecules that are linked to enhanced expression or stress factors during recombinant protein production.

Blaas, L., Musteanu, M., Eferl, R., Bauer, A., Casanova, E. (2009) Bacterial artificial chromosomes improve recombinant protein production in mammalian cells. BMC Biotechnol. 9:3
Kunert, R., Casanova, E. (2013) Recent advances in recombinant protein production. BAC-based expression vectors, the bigger the better. Bioengineered 4:4
Mader, A., Prewein, B., Zboray, K., Casanova, E., Kunert, R. (2013) Exploration of BAC versus plasmid expression vectors in recombinant CHO cells. Appl. Microbiol. Biotechnol. 97, 4049-4054
Mayrhofer, P., Kratzer, B., Sommeregger, W., Steinfellner, W., Reinhart, D., Mader, A., Turan, S., Qiao, J., Bode, J., Kunert, R. (2014) RMCE reference sites for efficient evaluation of different antibody expression levels, submitted
Turan, S., Zehe, C., Kuehle, J., Qiao, J., Bode, J. (2013) Recombinase-mediated cassette exchange (RMCE) − a rapidly−expanding toolbox for targeted genomic modifications. Gene 515, 1-27
Zambrowicz, B.P., Imamoto, A., Fiering, S., Herzenberg, L.A., Kerr, W.G., Soriano, P. (1997) Disruption of overlapping transcripts in the ROSA βgeo 26 gene trap strain leads to widespread expression of β−galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789-3794