Transposon mediated co-integration and co-expression of transgenes in CHO-DG44 cells
© Balasubramanian et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
Transposon systems mediate stable integration of exogenous DNA elements into a host cell genome, and have been successfully used in mammalian cells for the generation of stable cell lines. The piggyBac (PB) transposon system has been shown to have several advantages over the other transposon system available [1–3]. It has also been shown to generate stable cell lines at significantly higher frequency than the conventional transfections . Here, we investigated the efficiency of the piggyBac (PB) transposon to facilitate the co-expression of multiple artificial transposons, each bearing a single transgene and the puromycin resistance gene for selection. Green fluorescent protein (eGFP), red fluorescent protein (mKate), and a human IgG1 antibody were used as model proteins . The effect of the stringency of selection on pool productivity was determined with increasing concentrations of puromycin. The duration of selection necessary for the generation of recombinant cell pools was also tested by selecting for a period of either 5 or 10 days.
Materials and methods
Cells were transfected using linear 25 kDa polyethylenimine (PEI) (Polysciences, Eppenheim, Germany). All the transfections are done in a final volume of 10 mL. Transfected cultures were incubated at 37°C in 5% CO2 and 85% humidity with agitation at 180 rpm. The ratio of plasmid coding for Gene of Interest (GOI) to the plasmid coding for the transposase was kept constant at 9:1.
Generation of pools and clones
For the generation of stable pools, two days post transfection the cells were seeded at a density of 5 x 105 cells/mL in ProCHO5 and puromycin. The cells were placed under selection pressure for 5 or 10 days. In case of a 10 day selection period, the puromycin concentration in the cultures was replenished on day 7 post transfection by seeding at a density of 5 x105 cells/mL in fresh ProCHO5 with puromycin. For productivity analysis the cells were seeded at a density of 3 x105 cells/mL and analyzed at day four.
Stable clones were generated by limiting dilution of the pools. The productivity of the clones was analyzed after five days of culture.
A Guava EasyCyte microcapillary flow cytometer (Millipore) with excitation and emission wavelengths of 488 and 532 nm, respectively, was used to measure EGFP-specific fluorescence. The IgG concentration in the culture medium was determined by sandwich ELISA as previously described .
PB Transposition significantly enhances co-expression of multiple genes from stable pools compared to standard transfection
No significant differences were observed in the percentage of GFP positive cells and IgG productivity in cell pools generated by transposition with 5 or 10 days of selection. However, increased IgG productivity and % GFP-positive cells were observed with 10 days of selection in case of standard transfection (data not shown). This shows that a longer duration of selection pressure is necessary to generate pools by standard transfections compared to transposition. Furthermore, increased antibody productivity was observed with increasing puromycin concentration (Fig. 1).
PB transposition strongly improved clonal productivity
Distribution of clones based on their IgG productivity
Based on the above results we conclude that the piggyBac transposon system provides an efficient method for the co-integration of multiple genes. The use of the PB transposon system for the co-integration of multiple genes generates a higher frequency of high-producing clones than standard transfection. The GFP-expressing cell population was larger and the volumetric antibody productivity of the pools were higher with higher stringency of selection. A selection period with puromycin of only 5 days was sufficient for the generation of pools from transposon mediated transfection.
This work has been supported in part by the CTI Innovation Promotion Agency of the Swiss Federal Department of Economic Affairs (n. 10203.1PFLS-LS), in collaboration with the company ExcellGene SA in Monthey, Switzerland.
- Kahlig KM, Saridey AK, Kaja A, Daniels MA, George AL, Wilson MH: Multiplexed transposon-mediated stable gene transfer in human cells. Proc Natl Acad of Sci USA. 2010, 107 (4): 1343-8. 10.1073/pnas.0910383107.View ArticleGoogle Scholar
- Wilson MH, Coates CJ, George AL: PiggyBac Transposon-mediated Gene Transfer in Human Cells. Molecular Therapy. 2007, 15: 139-145. 10.1038/sj.mt.6300028.View ArticlePubMedGoogle Scholar
- Matasci M, Baldi L, Hacker DL, Wurm FM: The PiggyBac Transposon Enhances the Frequency of CHO Stable Cell Line Generation and Yields Recombinant Lines with Superior Productivity and Stability. Biotechnol Bioeng. 2011, doi: 10.1002/bit.23167Google Scholar
- Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM: Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng. 2001, 75: 197-203. 10.1002/bit.1179.View ArticlePubMedGoogle Scholar
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