Limits...
Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells.

Mayrhofer P, Kratzer B, Sommeregger W, Steinfellner W, Reinhart D, Mader A, Turan S, Qiao J, Bode J, Kunert R - Appl. Microbiol. Biotechnol. (2014)

Bottom Line: Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities.These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA.This new host is a prerequisite for cell biology studies of independent transfections and transgenes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology/Vienna Institute of BioTechnology (BOKU-VIBT), University of Natural Resources and Life Sciences (Vienna), Muthgasse 18, A-1190, Vienna, Austria.

ABSTRACT
Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.

Show MeSH

Related in: MedlinePlus

qPCR analysis of mRNA transcript level of two selected 3D6scFv-Fc- or 2F5scFv-Fc-producing RMCE clones cultivated in spinner flasks. Samples were taken at three different days and measured in two technical replicates. Total mRNA was reverse transcribed into cDNA and analyzed by qPCR using probes specific for the Fc sequence or β-actin used as an internal standard. Mean 2−ΔCp values were calculated based on differences of Cp values between β-actin and the Fc sequence. Error bars represent standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4231286&req=5

Fig5: qPCR analysis of mRNA transcript level of two selected 3D6scFv-Fc- or 2F5scFv-Fc-producing RMCE clones cultivated in spinner flasks. Samples were taken at three different days and measured in two technical replicates. Total mRNA was reverse transcribed into cDNA and analyzed by qPCR using probes specific for the Fc sequence or β-actin used as an internal standard. Mean 2−ΔCp values were calculated based on differences of Cp values between β-actin and the Fc sequence. Error bars represent standard deviation

Mentions: The four scFv-Fc-producing cell clones 1F11, 3B9, 1C3, and 3E5 chosen for routine cultivation in spinner flasks were analyzed for intracellular product formation and mRNA levels at three independent sampling days (indicated by arrows in Fig. 2c, d) to identify possible bottlenecks responsible for the differences in specific productivities of the two antibody variants. A single-parameter histogram at day 40 in spinner cultivation is depicted in Fig. 4a. A single peak of intracellular PE fluorescence (FL-2 signal intensity) was obtained from intracellular scFv-Fc for all individual sampling days (Online resource 4), as was expected for a RMCE guided exchange of transgenes. Comparing the median fluorescence intensity of two 2F5scFv-Fc and 3D6scFv-Fc-producing cell clones only minor variations were measured between the different sampling times indicated by small error bars. The median fluorescence intensity (MFI) of 3D6scFv-Fc-producing clones was a factor 1.5 higher than the 2F5scFv-Fc-producing clones (Fig. 4b). This difference does not necessarily reflect the 2-fold difference in specific productivities. Relative mRNA levels compared with beta-actin reference level were constant in all scFv-Fc-producing RMCE clones cultivated in spinner flasks (Fig. 5; Online resource 5). These data support the capacity of the new host cell line as a tool for the comparison of clones at a post transcriptional level.Fig. 4


Accurate comparison of antibody expression levels by reproducible transgene targeting in engineered recombination-competent CHO cells.

Mayrhofer P, Kratzer B, Sommeregger W, Steinfellner W, Reinhart D, Mader A, Turan S, Qiao J, Bode J, Kunert R - Appl. Microbiol. Biotechnol. (2014)

qPCR analysis of mRNA transcript level of two selected 3D6scFv-Fc- or 2F5scFv-Fc-producing RMCE clones cultivated in spinner flasks. Samples were taken at three different days and measured in two technical replicates. Total mRNA was reverse transcribed into cDNA and analyzed by qPCR using probes specific for the Fc sequence or β-actin used as an internal standard. Mean 2−ΔCp values were calculated based on differences of Cp values between β-actin and the Fc sequence. Error bars represent standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4231286&req=5

Fig5: qPCR analysis of mRNA transcript level of two selected 3D6scFv-Fc- or 2F5scFv-Fc-producing RMCE clones cultivated in spinner flasks. Samples were taken at three different days and measured in two technical replicates. Total mRNA was reverse transcribed into cDNA and analyzed by qPCR using probes specific for the Fc sequence or β-actin used as an internal standard. Mean 2−ΔCp values were calculated based on differences of Cp values between β-actin and the Fc sequence. Error bars represent standard deviation
Mentions: The four scFv-Fc-producing cell clones 1F11, 3B9, 1C3, and 3E5 chosen for routine cultivation in spinner flasks were analyzed for intracellular product formation and mRNA levels at three independent sampling days (indicated by arrows in Fig. 2c, d) to identify possible bottlenecks responsible for the differences in specific productivities of the two antibody variants. A single-parameter histogram at day 40 in spinner cultivation is depicted in Fig. 4a. A single peak of intracellular PE fluorescence (FL-2 signal intensity) was obtained from intracellular scFv-Fc for all individual sampling days (Online resource 4), as was expected for a RMCE guided exchange of transgenes. Comparing the median fluorescence intensity of two 2F5scFv-Fc and 3D6scFv-Fc-producing cell clones only minor variations were measured between the different sampling times indicated by small error bars. The median fluorescence intensity (MFI) of 3D6scFv-Fc-producing clones was a factor 1.5 higher than the 2F5scFv-Fc-producing clones (Fig. 4b). This difference does not necessarily reflect the 2-fold difference in specific productivities. Relative mRNA levels compared with beta-actin reference level were constant in all scFv-Fc-producing RMCE clones cultivated in spinner flasks (Fig. 5; Online resource 5). These data support the capacity of the new host cell line as a tool for the comparison of clones at a post transcriptional level.Fig. 4

Bottom Line: Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities.These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA.This new host is a prerequisite for cell biology studies of independent transfections and transgenes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology/Vienna Institute of BioTechnology (BOKU-VIBT), University of Natural Resources and Life Sciences (Vienna), Muthgasse 18, A-1190, Vienna, Austria.

ABSTRACT
Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.

Show MeSH
Related in: MedlinePlus