A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

Abstract

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

Fig. 1

Generation of a master cell line (MCL) harboring an inducible noncytopathic SFV vector expressing GFP. BHK cells were transfected with a plasmid constitutively expressing the rtTA2S-M2 transactivator linked to the hygromycin resistance gene (Hyg^R) by an IRES element (pPGK-TA-ires-Hyg, upper diagram). After selection with hygromycin, a clone was transfected with a plasmid containing the inducible noncytopathic SFV vector expressing GFP (i-ncSFV-GFP) and a neomycin resistance gene (Neo^R) (lower diagram). In this vector, the ncSFV sequence is under the transcriptional control of a minimal albumin promoter (Palb) fused to a tetracycline responsive element (tetO7), whereas the GFP sequence is located downstream of the SFV subgenomic promoter (Sg). Mutant LoxP sites were cloned upstream of the GFP sequence and downstream of Neo^R, respectively (LoxP1 and LoxP2). Cells were selected with neomycin, cloned by terminal dilution and analyzed in terms of inducibility, GFP expression, and absence of leakage, to obtain the i-ncSFV-LoxP-GFP MCL. PGK_p, phosphoglycerate kinase promoter; ncRep, non cytopathic SFV Rep; SV40, SV40 promoter; An, SV40 polyadenylation sequence; TKpolyA, Herpes simplex virus thymidine kinase polyadenylation sequence

Fig. 2

Kinetics of GFP expression in the MCL. MCL cells were incubated with 2 μg/ml of DOX at 33 or 37 °C and the presence of GFP-positive cells was analyzed at the indicated times by FACS (a) or with an inverted fluorescence microscope (b). Cells incubated without DOX were used as control. c Cell lysates were analyzed by Western blot with antibodies specific for GFP, Rep, and actin. Notice that for Rep panels, the part of the gel corresponding to 0–12 h was exposed five times longer than the one corresponding to 24–144 h (d). The MCL was plated at a density of 5 × 10⁵ cells per well in six-well plates. After 24 h, cells were incubated with DOX at the indicated concentrations and times. After 5 days, the percentage of GFP-positive cells was analyzed by FACS. Results in a and d are the mean ± SD values obtained from three independent experiments using passages 3, 5, and 7. Results shown in b and c correspond to cells analyzed in passage 4. Ind induced, Unind uninduced

Fig. 3

Stability of GFP expression. The MCL was passaged 50 times every other day with or without selective antibiotics (neomycin and hygromycin, Hyg/Neo). At the indicated passages, cells were incubated for 120 h with 1.25 µg/ml DOX (Ind.) or without DOX (Unind.) and GFP expression was evaluated by FACS (a) and Western blot with a specific antibody against GFP (b). ctrl stable cell line expressing GFP from an non-inducible RNA-based vector (ncSFV-pac2A-GFP) [21]

Fig. 4

Kinetics of GFP expression in a conventional inducible cell line. A BHK stable cell line constitutively expressing rtTA2S-M2 transactivator and containing GFP gene under the transcriptional control of tetO7-PAlb promoter was treated with 2 μg/ml of DOX or without DOX at 33 °C. Cell lysates were analyzed at the indicated times by Western blot using an antibody against GFP and actin. MCL(+), MCL incubated during 120 h with DOX

Fig. 5

Recombinase-mediated cassette exchange. a Schematic diagram of new cell lines’ selection process. In order to generate a stable cell line expressing a gene of interest (GOI), the MCL is cotransfected with a plasmid coding Cre recombinase (pCMV-CRE) and a shuttle vector harboring the GOI and the puromycin resistance gene pac (pShuttle-SFV-GOI). After Cre-mediated recombination the GFP-Neo^R cassette is substituted by GOI-pac, and cells become sensitive to neomycin and resistant to puromycin, which can be used to select the new cell lines. b Evaluation of transgene exchange using Tomato as GOI. The MCL was cotransfected with 1 µg of pCMV-CRE and 2 µg of the pShuttle-SFV-Tomato. After 24 h, puromycin was added to a final concentration of 5 µg/ml and selection was performed in batch until confluence. Once selected, the new cell line was induced with DOX at a final concentration of 1.25 μg/ml during 5 days, and evaluated for GFP and Tomato expression with a fluorescence microscope. EF1α elongation factor-1 alpha promoter

Fig. 6

Generation and characterization of stable cell lines expressing human cardiotrophin-1 (CT-1). a Diagram of pShuttle-SFV vector harboring the sequence of human CT-1 gene (pShuttle-SFV-CT). In this vector, CT-1 is fused in frame with the preprotrypsin signal peptide (SP) and a sequence coding for a six histidine-tag (His) at the amino-terminal end. b Analysis of CT-1 expression in the supernatant of clone CT1-B after DOX induction by Western blot with an antiserum specific for CT-1, using as control recombinant CT-1 produced in bacteria (recCT). c, d Analysis of CT-1 expression in three selected clones obtained after Cre-mediated cassette exchange. CT1 clones were passaged ten times in the absence of selective antibiotics and CT-1 expression was analyzed in supernatants collected 120 h after induction with DOX, or without induction, by Western blot as described in b (c) and specific CT-1 ELISA (d). e Cytotoxicity after protein expression was assessed by crystal violet staining at the indicated times using clone CT1-A

Fig. 7

Generation and characterization of stable cell lines expressing human Oncostatin M (OSM). a Diagram of pShuttle vector harboring the sequence of the OSM gene fused in frame with the preprotrypsin signal peptide (SP) and a histidine-tag (His) at the amino-terminal end (pShuttleSFV-OSM). b Analysis of OSM expression in the supernatant of clone OSM-A after DOX induction by Western blot with an antiserum specific for OSM, using as control recombinant OSM produced in bacteria (recOSM). c, d Analysis of OSM expression in three selected clones. OSM clones were passaged ten times in the absence of selective antibiotics and OSM expression was analyzed in supernatants collected 96 h after induction with DOX, or without induction, by Western blot as described in b (c) and specific ELISA (d). e Cytotoxicity after OSM expression was assessed by crystal violet staining at the indicated times using clone OSM-A. A clear cytopathic effect was observed only in cells incubated with DOX

Fig. 8

Kinetics of expression in cell lines adapted to growth in suspension. a The MCL was adapted to grow in suspension (S-MCL), cells were incubated with 2 μg/ml of DOX or without DOX at 33 °C and expression of GFP was analyzed in cell lysates at the indicated times by Western blot. MCL(+), adherent MCL cells incubated during 5 days with DOX used as control. b OSM-A cell line adapted to grow in suspension with serum (FBS), or without serum (w/o FBS) were induced with DOX as described, medium was replaced by new medium every 24 h and OSM expression in the supernatant was evaluated by specific ELISA at the indicated times. The curve represents mean values ± SD