Plasmid construction
Restriction and DNA modifying enzymes were purchased from New England Biolabs, Ltd (Pickering, Ont, Canada). Plasmids were constructed using standard molecular biology procedures and they were purified with the Qiagen plasmid Maxi Kit (Qiagen, Valencia, CA). To construct pMPG-IGF-E5, the coding sequence of IGF-1 was isolated by PCR from the murine cDNA of that gene (GenBank ID: BC012409, BF383724, ATCC, Manassas VA,) and primers 5'-ggatccggcaccatgtcgtcttcacacctc-3' and 5-ggtacctccacctgaaccggctgcttttgtaggcttc-3'. The PCR product was phosphorylated by treatment with T4 polynucleotide kinase and cloned into the unique PmeI site downstream of the CMV promoter of pMPGB34k, which was previously digested with BamHI to remove the light chain of the B43 antibody [32]. pMPGB43k is an expression vector that carries an enhanced version of the human CMV enhancer/promoter [20] and a hygromycin resistance gene. The DNA fragment encoding the His Tag and E5-Coil sequence [33, 34] was amplified by PCR using primers 5'-tattggtaccggcgggcaccatc-3' and 5'-gcgaggtaccgctagcttattacttctcaagtgctg-3'. The PCR product was digested with KpnI and cloned into the unique KpnI site downstream of the IGF-1 sequence previously inserted into pMPGB43k. The complete nucleotide sequence of IGF-E5 was confirmed by DNA sequencing. Plasmid pMPG-tPA, encoding tissue plasminogen (tPA) regulated by the CMV promoter and hygromycin resistance, was constructed by isolating the tPA cDNA by PCR from pETPFR (American Type Culture Collection). A first amplification was performed using primers AS1: 5'-gccgccaccatggatgcaatgaagaga-3' and AAS1: 5'-gttggatcctcacggtcgcatgttgtc-3' which inserted a Kozak consensus sequence at the 5' end and a unique BamH1 at the 3'end. A BamHI site was then added at the 5' end by performing a second PCR reaction with primers AS2: 5'-aacggatccgccgccaccatggatgca-3' and AAS1. The light chain of B43 antibody from pMPGB43k was then replaced with the PCR product by digestion with BamHI.
Cell culture
CHO cells adapted to serum-free suspension culture (CHO-SF, Invitrogen, Grand Island, NY), were grown in protein free chemically defined medium for CHO cells (CD-CHO medium, Invitrogen) supplemented with 1× HT supplement (Invitrogen), 4 mM glutamine and 50 μg/ml dextran sulfate (MW: 500 000, Amersham Pharmacia Biotech, Uppsala, Sweden). 293SF [25] were grown in low-calcium-SFM (LC-SFM, Invitrogen, Grand Island, NY) supplemented with 1% fetal bovine serum (HyClone, Logan, Utah). Unless mentioned otherwise, all the cells were grown at 37°C in the presence of 5% CO2.
Transfection and stable clone production
CHO-SF and 293SF were transfected by electroporation in the presence of linearized plasmid using a BTX T820 electrosquare porator (Genetronics, San Diego, CA) according to the manufacturer's recommendations. For the clone isolation using the "standard method", after electroporation, the CHO-SF were diluted into growth medium and incubated overnight in 6-well plates. The next day, they were separated into 96-well plates (1000 and 3000 cells/wells) in growth medium supplemented with 600 μg/ml hygromycin B (Invitrogen). Resistant colonies, isolated from plates in which less than 30 wells contained resistant colonies to maximize the odds of isolating a true clone, were then transferred to 24-well plates in the presence of hygromycin B. Supernatant from confluent 24-well plates were analyzed by dot blot for the presence of IGF-E5. To generate clones using the FLSSM method (see below), after electroporation, a pool of stable transfectants was generated by growing the cells in the presence of 600 μg/ml (CHO-SF) or 25 μg/ml (293SF) hygromycin B.
Cell plating in semi-solid medium
Immediately before plating in semi-solid medium, cells were dispersed in a 35 mm Petri dish through a fine microtip (Costar, Bethesda, MD) and observed under the microscope. The process was repeated until only single cells were observed. CHO-IGF-E5 and CHO-tPA stable cell pools were mixed in medium containing Clonematrix methylcellulose, XL Reagent (Genetix, Boston, MA) and CDCHO-AGT 2× (Invitrogen), at a 1× final concentration and supplemented with 8 mM L-glutamine (Invitrogen) and fluorescent antibody (see below). 293SF-IGF-E5 stable cell pools were mixed in medium containing Clonematrix, 2× LC-SFM medium (1× final concentration), 1%FBS (HyClone) and 4 mM L-glutamine. The cells (CHO and 29SF) were agitated and vortexed at medium speed to ensure uniform distribution of all components before plating. They were then seeded in 24-well dishes (Sarstedt, Newton, NC) or 60 mm Petri dishes (Greiner, Monroe, NC), made of untreated plastic, at densities of 500, 1000 and 2000 cells/m. After plating, the cells were observed under the microscope to verify they were single and well dispersed inside the dish.
Cell labeling and colony picking
Anti-His Tag-FITC antibody (Abcam, Cambridge, MA) was used at 4 μg/ml to detect IGF-E5 secreting cells in semi-solid medium. The anti-tPA (Cedarlane, Hornby, Ont) antibody was first dialyzed on a microcon YM-50 spin filter (BioRad, Missisauga, ON) against PBS (HyClone), and conjugated to Alexa488 fluorochrome with a Microscale Protein Labeling Kit (Molecular Probes, Eugene OR) according to the manufacturer's recommendations. The labeled antibody was used at a concentration of 1 μg/ml to detect tPA-secreting cells. Positive cells and colonies were picked either with a Quixell micromanipulator (Stoelting, Wood Dale, IL) using 50 μm glass capillaries, or with a CellCelector™ colony picker (Aviso, Jena, Germany) equipped with the methylcellulose tool. For Quixell picking, the most fluorescent cells were determined visually. For CellCelector picking, the plates were scanned at 10× magnification and the brightest objects were determined using the included analySIS software (now called Cell D). Single cells and colonies were deposited into 96-well dishes (Corning, Lowell, MA). Images of fluorescently labeled cells were taken on a Leica DMIL inverted microscope using a color 3CCD cooled camera (Optronics, Goleta, CA) or a monochrome Retiga Exi (QImaging, Surrey, BC) and Openlab software (Improvision, Coventry, England).
Analysis of protein production by dot blots and spot blots
For dot blot analysis, the culture supernatant was diluted 5 times with distilled water. The samples were denatured by adding an equal volume of 2× lysis buffer (0.125 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol and 0.1% bromophenol blue) and heated at 95°C for 5 min. The samples were spotted on a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences Buckinghamshire, UK) under vacuum on a dot-blot apparatus (Bio-Rad Laboratories Canada Ltd., Mississauga, ON). The membrane was incubated with an anti-His Tag antibody (Serotec, Oxford, UK) followed by a horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Biosciences). The signal was revealed by chemiluminescence using the ECL Western Blotting detection reagents (Amersham Biosciences). For spot blot analysis [35] duplicate or triplicate volumes of one microliter were spotted on nitrocellulose membranes (Hybond-ECL) with a precision P2 micropipette (Eppendorf, New York, NY). Quantitative spot blots were made from batch samples, from clones seeded at 5 × 105 cells/ml and grown for 3 days (CHO-SF) or 5 days (293SF) under identical conditions at 30°C. At least three dilutions were spotted for each clone to ensure that detected values would fall in the linear domain of the standard curve. IGF-E5 blots were treated with an anti-His Tag antibody (Serotec) followed by an anti-mouse conjugated to Alexa488 (Invitrogen). tPA blots were treated with a sheep anti-tPA primary antibody (Cedarlane) followed by a rabbit anti-sheep antibody conjugated to Cy3 (Jackson, Immunoresearch Labs, West Grove, PA). Protein standards (purified IGF-E5 or tPA (Calbiochem, San Diego, CA)) were spotted in duplicate or triplicate serial dilutions on each blot. The blots were scanned on a Typhoon Trio+ fluorescence imager (Amersham Biosciences) and analyzed by ImageQuant TL software (Amersham Biosciences). The data was transferred to Excel spreadsheets for standard curve plotting and result interpolation.
Analysis of protein production by Western blot
Stable CHO transfectants (5.0 × 105) were resuspended into 1 ml of growth medium in 24-well plates. The next day, the plates were transferred to an incubator at 30°C and incubated at this temperature for up to 14 days. At selected time points, an aliquot of growth medium was mixed with an equal volume of lysis buffer 2× supplemented with 5% β-mercaptoethanol. The samples were heated for 5 min at 100°C and separated on a SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and the presence of the IGF-E5 was demonstrated using anti-His Tag antibody by chemiluminescence as described above.
Purification of IGF-E5
Actively growing CHO cells from clone 4-B4 (1.0 × 106/ml) in a T-Flask were incubated at 30°C for 7 days. After separation of supernatant and cells by centrifugation, Complete protease inhibitor (Roche Diagnostics, Mannheim, Germany) and a final concentration of 10 mM imidazole were added to the supernatant. The supernatant was loaded onto a 5-ml HisTrap column (Amersham Biosciences) using a peristaltic pump and the bound IGF-E5 was eluted with increasing concentrations of imidazole (10 to 200 mM). The purified IGF-E5 was dialyzed against PBS and concentrated on a centrifugal concentrator (Centricon YM-10, Millipore Corporation, Bedford, MA). After determining the protein concentration with the DC protein assay (Bio-Rad laboratories, Hercules, CA), the purified IGF-E5 was stored at -80°C in 5% glycerol. The purity of the protein was evaluated by SDS-PAGE followed by staining with Coomassie Fluor™ Orange protein gel stain (Molecular Probes, Eugene OR) and analyzed on a Typhoon trio+ scanner (Amersham Biosciences). The nature of the purified IGF-E5 was confirmed by Western blot (as described above) using as primary antibody an anti-His Tag antibody or a goat anti-IGF-1 antibody (USBiological, Swampscott, MA).
Correlation of production and fluorescence in FLSSM
Seven CHO-IGF-E5 clones of various production levels (as per initial spot blot screening) were seeded at 1.0 × 104 cells/ml in semi-solid medium containing anti His Tag-FITC antibody in duplicate wells of a PetriWell24 plate (Genetix) and, in parallel batches at 1.0 × 106 cells/ml in liquid growth medium. Duplicate wells of a 24-well dish (Sarstedt, Montréal, QC) were incubated at 30°C for 3 days. Samples were clarified by centrifugation and analyzed by quantitative spot blot as described above. Semi-solid medium plates were scanned on a CellCelector™ (Aviso). The product of area and mean fluorescence for all the colonies above background threshold was determined with the Cell D software (Aviso/Olympus) and summed for each separate clone using an Excel spreadsheet. The results were normalized to the brighest clone (L). Quantitative spot blot results for batch production were normalized to the highest producer (L).