Molecular cloning
The sequences of the primers used for cloning expression plasmids are shown in Additional file 1: Table S1. The backbone vector, pBL-2, was obtained by two stages of inverted PCR using long adapter primers and the pUC18 plasmid as a template. Non-functional parts of the plasmid including the pLac promoter and the LacZ gene were removed. Inverted PCR was performed as described previously [12]. Oligonucleotides and PCR reagents were from Evrogen, JSC (Moscow, Russia). PCR products were purified from 1% agarose gels by the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). T4 polynucleotide kinase and MalI restriction endonuclease (Sibenzyme, Novosibirsk, Russia) were used. T4 DNA ligase (Fermentas, Vilnius, Lithuania) was used for inverted PCR product circularization. The Escherichia coli TOP10 strain (Invitrogen, Carlsbad, CA) was used for cloning. Plasmids were isolated with a GeneJet Plasmid Purification Kit (Fermentas, Vilnius, Lithuania).
The pAL-EBV plasmid, containing a fragment of a concatemer of EBV terminal repeats, as described previously [5] and nearly undistinguishable from the human herpes virus 4 strain K4123-MiEBV sequence [GenBank: KC440852.1] was made from synthetic oligonucleotides cloned into a pAL-TA (Evrogen) vector. The ORF encoding mouse DHFR was obtained by PCR using pOptivec-TOPO linearized vector (Invitrogen) as a template. The fragment encoding the attenuated encephalomyocarditis virus (EMCV) IRES was obtained from the pOptivec-circ plasmid (self-ligated pOptivec-TOPO) by restriction. These fragments were cloned into the pBL-2 plasmid via assembly of two different intermediate constructs, PBL-2-ID and PBL-2-ID-EBV. DNA modification enzymes for routine molecular cloning were obtained from Fermentas or Sibenzyme.
Construction of p1.1 vectors
Fragments corresponding to the upstream and downstream flanking regions (8532–12603 and 14545–18794 sequences of [GenBank:AY188393]) of the CHO elongation factor 1 gene were obtained by PCR using CHO DG44 cell (Invitrogen) genomic DNA as a template. The modular assembly cloning technique used herein is described in detail elsewhere [13].Assembled CHO genomic regions were cloned into the intermediate plasmids, PBL-2-ID and PBL-2-ID-EBV, resulting in p1.1(EBVTR-) and p1.1 expression vectors, respectively (Figure 1).
Construction of p1.2 vectors
p1.2-Mono, the intermediate backbone plasmid for expression vectors bearing antibiotic resistance genes was obtained by removal of the region containing the EMCV IRES and the DHFR ORF from the p1.1 expression vector. Plasmid pAL-3CH123, containing first three modules of the downstream flanking region of the EEF1A was used as the source of the donor DNA insert fragment, replacing the deleted IRES and DHFR area, so both flanking regions of the EEF1A remained unaltered (Figure 2). Antibiotic resistance genes and the SV40 promoter and terminator regions were obtained by amplification with adaptor primers, using pcDNA3.1/Hygro, pcDNA3.1(+), and self-ligated pcDNA4/HisMax-TOPO (Invitrogen) as PCR templates. Antibiotic resistance cassettes were sub-cloned into T-vectors and then transferred into the p1.2-Mono backbone by restriction-ligation resulting in p1.2-Hygro, p1.2-Neo and p1.2-Zeo. A DNA fragment encoding eGFP and a consensus Kozak sequence (GCCGCCATGG) [14] was obtained by PCR with adaptor primers and the pEGFP-C2 plasmid (Clontech, Mountain View, CA) as a template and then cloned into the polylinker area of p1.1 and p1.2 vectors, thereby resulting in p1.1(EBVTR-)eGFP, p1.1eGFP, p1.2-HygroeGFP, p1.2-NeoeGFP and p1.2-ZeoeGFP expression plasmids.
Purified plasmids for transfection and the control plasmid pEGFP-N2 (Clontech) were prepared using an EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA). For stable cell line generation all plasmids except p1.2-HygroeGFP were linearized by restriction with PvuI by cutting inside the ampicillin resistance gene bla sequence. The plasmid p1.2-HygroeGFP was restricted with BspHI, which introduced two breaks near the bla gene.
Cell culture
A DHFR-negative CHO DG44 cell line (Invitrogen) was cultured in shaking flasks in the chemically defined medium, CD DG44 (Invitrogen), supplemented with 0.18% Pluronic F-68 (Invitrogen) and 4 mM L-glutamine (Invitrogen). The cells were passaged 24 h before transfection. For direct colony generation in 96-well culture plates, transfection was performed using Fugene HD reagent (Promega), containing 60 μg of DNA and 180 μl of the reagent per 15 millions of cells in 30 ml of the above medium. Plasmids p1.2 were transfected by electroporation in Gene Pulser Electroporation Buffer (Bio-Rad, Hercules, CA) using a cuvette with a 4 mm gap with 7.5 million cells and 15 μg of linearized DNA for each transfection. Cells were counted by trypan blue exclusion and fluorescence microscopy at 48 h post-transfection. For direct generation of colonies, transiently transfected cell cultures were transferred into CHO-A culture medium (Invitrogen) lacking hypoxanthine and thymidine (HT), and seeded at 5000 cells/well in the culture plates. Cells were grown undisturbed for 14 days and inspected by fluorescence microscopy. The medium was changed and the plates were cultivated for 5–10 additional days until the first 10% of the wells containing colonies became confluent.
To generate stably transfected cell populations using p1.1eGFP and p1.1(EBVTR-)eGFP plasmids, transiently transfected cultures were transferred to OptiCHO medium (Invitrogen) lacking HT, and thereafter cultivated in shaking flasks with medium exchange every 3 days until the cell viability increased to 85% (approximately 22–27 days of cultivation).
MTX-driven target gene amplification in culture plates was performed by seeding the cells from stably transfected cell populations into 96-well culture plates at a density of 5000 cells/well in the CHO-A culture medium, supplemented with 0, 50, 100, 200, 400 or 800 nM MTX. Three plates were used for each concentration of MTX. The cells were grown undisturbed for 14 days, after which the plates were inspected by microscopy and the culture medium was changed every 4 days until the first 10% of wells in each plate became confluent. Plates were screened again by fluorescence microscopy, and cells from the 16 brightest wells from each plate were transferred into a 48-well plate, grown to confluence, and then transferred into 24-well plates. Colonies lacking normal proliferation speeds or attached to the surface of the plates too tightly for dislodging by pipetting were discarded. Cells from the eight brightest wells for each MTX concentration were dislodged from their plates, lysed as described below, and then used to determine eGFP levels. Six randomly picked colonies, obtained in the presence of 400 and 800 nM MTX, were transferred into a 6-well plate and grown with shaking in OptiCHO medium with passages made every three days for 60 days. Samples for eGFP level determination were collected every second passage.
Target gene amplification for polyclonal cell populations was performed for the suspension culture of CHO DG44 cells, stably transfected by the p1.1eGFP plasmid in presence of 50 nM MTX, as described above. Concentration of the MTX in the culture medium was increased by two-fold steps, each after two consecutive passages, until the cell viability decreased below 85%. Resulting culture, obtained in presence of 0.8 μM MTX, was split into four flasks, supplemented by 0.8; 1.6; 3.2; 6.4 μM MTX and cultured until the cell viability returned to at least 85% (7–12 days).Generation of polyclonal cell populations involving transfected p1.2 plasmids were performed by seeding transiently transfected cells in 6-well culture plates, using 1 million of viable cells per well in 5 ml of DG44 medium, supplemented with the corresponding antibiotic, or 5 ml of OptiCHO medium with 200 nM MTX for control transfections using p1.1 plasmids. The concentrations of the antibiotics used are shown in Figure 3. Plates were cultivated with shaking until the cell viability returned to at least 85% (20 days), after which the medium was changed every 4 days.
Determination of eGFP concentrations in cell lysates
Cell culture samples containing approximately one million of cells were centrifuged and the cell pellets were resuspended in phosphate buffered saline and recentrifuged. The washed cell pellets were resuspended in 0.1 ml of lysis solution containing 150 mM NaCl, 50 mM Tris–HCl at pH 7.5, 1% Triton X-100, a protease inhibitor cocktail (Sigma, St. Louis, MO), and then incubated for 30 min on ice with stirring. Cell debris was removed by centrifugation. The concentration of eGFP in the lysate from the H-4 cell population (Figure 3) was measured by spectrophotometry at a wavelength of 488 nm using a molar extinction coefficient of 55,000 M−1 · cm−1 and an eGFP molecular weight of 32.7 kDa [15]. The fluorescence intensity of eGFP in all of the lysates was measured along with the serially diluted calibration samples, which were prepared from the H-4 lysate containing a known concentration of eGFP. Total protein concentration in the lysates was measured by the Bradford method with bovine serum albumin as a standard.
FACS analysis and quantitative PCR
Undiluted cell culture samples were subject to FACS FC 500 (Beckman Coulter, Krefeld, Germany) analysis at an emission at 488 nm and detection through a 530/40-nm bandpass filter. At least 10,000 individual cells were counted for each sample analysed. Quantitative PCR analysis of the expression plasmid copy numbers in the genomes of stably transfected cells was performed using an iCycler iQ thermocycler (Bio-Rad) and qPCRmix-HS SYBR (Evrogen) reaction mixture with the primers shown in Additional file 1: Table S2. The highly purified p1.1eGFP plasmid was used as a quantity calibrator using five different concentrations for each determination performed in triplicate. PCR was performed three times with three to four replicates for each sample. Genomic DNA was extracted from cells with a Genomic DNA Purification Kit (Fermentas) and quantified using a Qubit fluorometer (Invitrogen) and the dsDNA HS kit (Invitrogen). Quantity calibrator plasmid was used as the external standard for the quantification of genomic DNA samples by fluorometry.