A GFP-based method facilitates clonal selection of transfected CHO cells.
Summary (2 min read)
Introduction
- A key step in the generation of cell lines for the efficient production of recombinant proteins is the selection of high producer clones following transfection and selection [1].
- The process of randomly picking a large number of clones produced, e.g. by limited dilution, and assaying them individually by ELISA is tedious, time consuming, and reportedly not always very effective [2,3].
- Moreover, this method does not account for differences in either cell density or media volume between wells.
- The enhanced green fluorescent protein (EGFP) has been suggested in the past as co-expressed indicator protein for applications in FACS-based cell separation, but also for the screening of cultures in multiwell plates [8-10].
- The titres are assumed to be linked.
Materials and Methods
- Materials 96-well plates used for screening were from Nunc, Wiesbaden, Germany (96 F TC Nunclon) and BD-Falcon, Heidelberg, Germany (Microtest 96).
- Chemicals were from established suppliers such as Sigma Aldrich and used as received.
- High quality water was produced by a Millipore unit.
- Cell culture Chinese Hamster Ovary cells (cell line CHO-K1 (CCL-61, ATCC)) were maintained in growth medium (R10: RMPI 1640 medium supplemented with L-glutamine (2 mM), penicillin/streptomycin (0.1 mg/mL) and 10 % foetal calf serum) in an atmosphere of 5.0 % CO2 and at 37 °C (incubator: Forma Steri-Cult or Forma Direct-Heat, ThermoFisher Scientific, Dreieich, Germany).
- Prior to the spinner (IIBS, Chur, Switzerland) experiments, the adherent cells were adapted to cultivation in suspension.
Plasmid construction
- The coding regions of the human growth factors VEGFA and IGF1A were amplified by RTPCR as follows, using total human RNA from Saos-2 and HEK293 as template.
- The PCR products were purified and subcloned into pGEMT-Easy (Promega, Mannheim, Germany).
- After 48 h, the cells were collected by trypsinisation and the transfection efficiency was roughly estimated via the EGFP fluorescence by flow cytometry in a Cytomics FC500 (Beckman Coulter, Krefeld, Germany).
- The basis for this method is a dilution of the cell suspension to a point were statistically less than one cell per well (here 0.6) are plated.
- The concentration of hVEGFA and hIGF1A in cell culture supernatants were determined using the specific ELISA development kits from Peprotech (London, UK) and from R&D systems (Wiesbaden, Germany) according to the manufacturer’s instructions.
Results and discussions
- In the pEGFP-N1-based gene constructs used here, the genes encoding for the recombinant target protein and for EGFP are linked by an internal ribosome entry site (IRES).
- Moreover, the use of pEGFP-N1 for transgene expression besides good product titres also makes possible the screening strategy developed in this contribution Determination of optimal screening parameters.
- Both examined types of transparent microplates exhibited a rather high background fluorescence, if compared to values given by the suppliers for the special fluorescence plates.
- The detected fluorescence values were in the range of BFplate, indicating that the contribution of the non-transfected cells to the measured fluorescence is low and does not depend on the number of cells per well.
- Nunc plates were thus used in the subsequent measurements.
Screening for high producers
- In order to test the method for clone screening, CHO cells were transfected with pEGFPhVEGFA or pEGFP-hIGF1A.
- Subsequently, the transfected cells were cultivated for two weeks under selection pressure before being subjected to limited dilution into 96-well plates.
- The intensity of the EGFP fluorescence was in the same range for all the investigated clones, the quantity of secreted hVEGFA was ten-fold higher than that of hIFG1A.
Conclusions
- The use of a recombinant bicistronic plasmid to express the protein of interest along with EGFP allows for a fast screening for “high producers” via the EGFP fluorescence.
- The screening can be performed in standard transparent microtitre plates and less than 3000 EGFP expressing cells can be detected.
- The new screening method allows an efficient detection of good producer clones at an early stage of the process development.
- As it does not rely on fluorescence-activated cell sorting, nor does it require the availability of an antibody specific for the therapeutic protein to be expressed, it can be easily implemented in any laboratory and stage of the cell line development process.
- Eva Weiss and Nicole Andersen provided technical support.
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...The recombinant cell line 4D6 was derived from the CHO-K1 parent cell line by transfection with phBMP2-EGFP, using PEI as transfection agent, followed by G418-selection and EGFP-based screening/adaptation to suspension culture as previously described [30]....
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...EGFP expression was measured as previously described by flow cytometry [31] or spectrophotometrically [30]....
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..., 2012) and growth factors (Freimark et al., 2010; Meng et al., 2000), thus enabling easy, rapid identifications of high producers....
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...Often, there is a high correlation between clone production of GFP and (easy-to-express) protein of interest including MAb (Davies et al., 2013; Kim et al., 2012) and growth factors (Freimark et al., 2010; Meng et al., 2000), thus enabling easy, rapid identifications of high producers....
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