Experimental workflow and vector designs
One way to validate the all-in-one reporter system is through transient or stable transfection of mammalian cells. Within this framework, the schematics in Fig. 1 illustrate the overall workflow of this study and feature the characteristics of promoters. To summarize, the study involves the engineering and validation of a reporter system containing nine common promoters with functionally and structurally diverse attributes: three viral promoters (CMV-mIE, SV40, and HSV-TK), three human promoters (EF1α, EFS, and UBC), one synthetic promoter (CAG), one mouse promoter (PGK), and one Chinese hamster promoter (CHEF1α) (Fig. 1C,D). The vector collection also features an empty control reporter lacking any variable promoter sequence (NP: no promoter). To experimentally verify the dual luciferase reporter system, first we comparatively investigated the transcriptional regulatory activities of the promoter panel in a transient transfection setting of CHO cell variants and HEK-293T cells (Fig. 1A, Experimental setting 1). Next, we reconstructed the reporter system to coexpress eGFP and tdTomato fluorescent reporters, with a focus on a subset of promoters that were selected and prioritized from the prior analysis (Fig. 1A, Experimental setting 2). Finally, we demonstrated that with further engineering the dual luciferase reporter system could be employed to quantify the strength of regulatory sequences in stable transfectants of CHO-DG44 suspension cells (Fig. 1B). Of particular relevance, CHO-DG44 cell line exhibits homozygous deletion of the entire dihydrofolate reductase (DHFR) locus, a feature routinely exploited for auxotrophic selection and transgene copy number amplification13,14,15,16. Finally, a detailed schematic of the modular vector backbones used throughout this study is rendered in Fig. 1E.
Experimental workflow of the study, promoter characteristics and vector designs. (A,B) Schematic representation of the workflow: Promoter strengths in the transient expression context were quantified using dual luciferase assay and flow cytometry analysis. (A) All nine promoters were compared based on luciferase activity in CHO cell variants and HEK-293T cells (Experimental setting 1). To corroborate the dual luciferase reporter findings, one weak and three relatively strong promoters were cloned into the dual fluorescence reporter system and further investigated by flow cytometry analysis (Experimental setting 2). (B) The activity of five relatively strong promoters selected and prioritized from (A) was evaluated in stable transfectants of CHO-DG44 suspension cells by dual luciferase assay. Further experiments were performed to analyze the expression of Fluc and Rluc genes, as well as the copy number of Rluc and DHFR genes. (C) The list of well-known constitutive promoters tested herein. Color codes represent the origin of promoters. (D) The table depicts promoter lengths. (E) Representative vector maps of all-in-one reporter systems. In the dual luciferase reporter system, the variable promoter (CMV-mIE) was replaced by SV40, HSV-TK, EF1α, EFS, UBC, PGK, CHEF1α and CAG promoters. In the dual fluorescent reporter system, the variable CMV-mIE was replaced by SV40, HSV-TK and CHEF1α promoters. For stable expression analysis, the constructs with CMV-mIE, SV40, UBC, CHEF1α and CAG promoters were engineered to coexpress Rluc and DHFR genes separated by an IRES sequence, creating a bicistronic expression cassette. The schematics in (E) are created with BioRender.com.
Transient transfection of the dual luciferase reporter system
Transient transfection technique in which the foreign DNA does not integrate into the host genome enables simple, quick, and cost-effective analysis of transgene expression17. This experimental scheme would allow for rapid validation of the bioluminescence reporter toolkit and provide an early assessment of promoter activities (Fig. 1A). To this end, we individually transfected CHO-WT, CHO-DG44 adherent, and CHO-DG44 suspension cells with the vector panel using an optimized protocol with a 1:3 mass-to-volume ratio of plasmid DNA to Lipofectamine 3000. We achieved comparable transfection efficiencies in CHO-WT and CHO-DG44 adherent cell lines with pEGFP-N1, a well-known plasmid that expresses the eGFP fluorescent protein. However, CHO-DG44 suspension cells had a lower efficiency with this protocol (data not shown). At 48 h post transfection, we performed the dual luciferase reporter assay to quantify the activities of each promoter. We estimated and correlated promoter strengths by calculating the ratio of Fluc to Rluc bioluminescence signals. Of the nine promoters, the CMV-mIE promoter displayed the highest activity (normalized to 100) in CHO-WT cells, followed by CAG (58.3), UBC (32.3), EF1α (28.5), CHEF1α (28.1), and SV40 (27.1) promoters, while the activities of PGK (22.7), EFS (18.28), and HSV-TK (12.7) promoters were relatively weaker (Fig. 2A). Intriguingly, the overall activity profile of other promoters was lower in CHO-DG44 adherent and CHO-DG44 suspension cells as opposed to CMV-mIE. In particular, the promoter activities in CHO-DG44 adherent cells were as the following: SV40 (8.5), CAG (7.9), UBC (7.8), EF1α (7.2), CHEF1α (7.1), PGK (5.2), EFS (5.1), and HSV-TK (2.4) (Fig. 2B). Similarly, the strongest promoter in CHO-DG44 suspension cells was CMV-mIE, which was followed by SV40 (18.8), UBC (17.8), CAG (15.6), EF1α (14.8), CHEF1α (13.7), EFS (9.9), PGK (8.7), and HSV-TK (4.3) promoters (Fig. 2C). The Fluc signal of the NP construct was nearly undetectable, proving that the Fluc expression was selectively driven by the variable promoters. These results indicate that under transient transfection conditions the CMV-mIE promoter is the strongest in CHO cell variants. In contrast, the remaining promoters deliver less and slightly variable activities in CHO-DG44 adherent and CHO-DG44 suspension cells when compared to CHO-WT data, an important finding that warrants further investigation.
Analysis of promoter strength using the dual luciferase reporter system in transient expression settings. The activities of nine promoters was evaluated by dual luciferase assay at 48 h post-transfection. Fluc expression is driven by the variable promoter and enables the systematic comparison of promoter activities, while the Rluc signal is an internal control regulated by the invariable CMV-mIE promoter. The measurement of promoter strength based on the ratio of Fluc to Rluc signals was investigated in (A) CHO-WT, (B) CHO-DG44 adherent, (C) CHO-DG44 suspension, and (D) HEK-293T cell lines. The CMV-mIE promoter containing vector exhibited the highest value which was normalized to 100 in all cell lines. (E) Cross comparison of promoter activities across cell lines was examined by setting the CMV-mIE promoter as a standard. The relative activity in HEK-293T cell line was set at 100.
Next, we sought to establish additional evidence demonstrating that the dual luciferase reporter system can have a widespread utility in mammalian cell cultures. To that purpose, we investigated the relative strength of promoters in HEK-293T, a highly transfectable variant of HEK-293 cell line that expresses a temperature-sensitive mutant of the SV40 T antigen. In essence, the human embryonic kidney 293 (HEK-293) cell line and its variants are particularly attractive mammalian host systems for recombinant protein production18,19. In close agreement with the CHO data, we found that the CMV-mIE promoter had the greatest activity (Fluc/Rluc) in HEK-293T cells. This was followed by CAG (19.0), EF1α (6.0), CHEF1α (5.1), PGK (4.7), EFS (3.8), UBC (3.2), and HSV-TK (2.1), SV40 (1.1) promoters. As expected, the NP construct had no detectable Fluc activity (Fig. 2D). Lastly, we set out to provide a comparative analysis of Fluc/Rluc ratios across cell lines, using the CMV-mIE promoter activity as a reference. In this case, HEK-293T cells exhibited the strongest activity (normalized to 100), while the lowest activity was observed in CHO-DG44 suspension cells (Fig. 2E). Using this information, the remaining promoters may be cross compared across four different cell lines. Taken together, these results successfully validate the all-in-one dual luciferase reporter system and offer compelling insights that this single vector toolkit can be utilized in transient expression settings for rapid and systematic assessment of promoter strength and perhaps other regulatory sequences in CHO and HEK-293 cell lines, and their variants. This would most likely be extended to other mammalian host cell lines.
Dual fluorescence reporter system in transient expression setting
After verifying the utility of the dual luciferase reporter system in CHO cell variants and HEK-293T cells, we attempted to expand the engineering capacity of the modular vector toolkit. To that goal, we specifically remodeled the dual luciferase vector backbone into an all-in-one dual fluorescence reporter system through extensive molecular cloning, substituting the Fluc and Rluc genes with eGFP and tdTomato coding sequences, respectively. Just like the dual luciferase reporter constructs, the new all-in-one system also provides simultaneous expression of the fluorescent reporter genes. Specifically, while tdTomato expression is controlled by the invariable CMV-mIE promoter, eGFP expression is dictated by the variable promoters, specifically CMV-mIE, SV40, CHEF1α, and HSV-TK (Fig. 1E). To examine promoter activities, we transiently transfected the vector panel into CHO-WT, CHO-DG44 adherent and CHO-DG44 suspension cells. At 48 h post-transfection, we performed flow cytometry analysis to quantitatively monitor eGFP and tdTomato reporter signals. Again, much like the previous calculations, the ratio of total eGFP-positive cells to total tdTomato-positive cells (eGFP/tdTomato) was expressed as a surrogate parameter of promoter strength. In consequence, we found that CHO-WT cells transfected with the CMV-mIE promoter-containing vector had the greatest ratio for eGFP/tdTomato (0.92), followed by CHEF1α (0.75) and SV40 (0.57) promoters, while the HSV-TK promoter exhibited the lowest value (0.45) for eGFP/tdTomato ratio (Fig. 3A,D). When compared to CHO-WT cells, CHO-DG44 adherent cells displayed the same order of promoter activities however with slight differences in eGFP/tdTomato ratios. Specifically, the CMV-mIE promoter had a ratio of 1.12, followed by CHEF1α (0.86), SV40 (0.73), and HSV-TK (0.50) promoters (Fig. 3B,D). Despite having a significantly lower transfection efficiency, CHO-DG44 suspension cells also exhibited the same order of promoter strength, with the eGFP/tdTomato ratios being 0.89 for CMV-mIE, 0.58 for CHEF1α, 0.51 for SV40, and 0.38 for HSV-TK promoters (Fig. 3C,D). In summary, we conclude that the dual fluorescent reporter system may offer an alternative to the bioluminescence based reporters for monitoring the strength of regulatory sequences. Besides, these data fortunately corroborate the versatility of the vector backbone, implying that further modifications could be tailored to suit individual requirements or specifications.
Cross validation of bioluminescence based results and analysis of promoter strength using the dual fluorescence reporter system in transient expression settings. Promoter strength was investigated by flow cytometry to verify bioluminescence based analyses while also demonstrating that the reporter system can function with fluorescence signals. The vector panel carrying CMV-mIE, SV40, HSV-TK and CHEF1α promoters was transiently transfected into CHO cell variants. At 48 h post-transfection, flow cytometry was conducted to evaluate promoter activities on (A) CHO-WT, (B) CHO-DG44 adherent and (C) CHO-DG44 suspension cells. (D) Similar to the dual luciferase reporter system, the ratio of eGFP/tdTomato fluorescence signals provides a systematic and quantitative comparison of the promoters. The strength of each promoter was expressed as the ratio of eGFP positive cells (Q1 + Q2) divided by the percentage of tdTomato positive cells (Q2 + Q3).
Dual luciferase reporter system in CHO-DG44 stable polyclonal cell pools
CHO host cell lines can deliver robust and sustained transgene expression, making them the most preferred mammalian cell system for recombinant protein production. In light of this, we explored whether the dual luciferase reporter system validated in transient transfection experiments could be readily applicable and adaptable to analyzing regulatory sequences in stable expression settings. As portrayed schematically in Fig. 4, we conducted a series of experiments in high-density stable transfectants of CHO-DG44 suspension cells, in which the constructs had successfully integrated into their genome. Basically, we selected and prioritized five dual luciferase reporter constructs which displayed relatively strong promoter activity in transient transfection assays (CMV-mIE, SV40, UBC, CAG, and CHEF1α) and modified them accordingly. Using an overlap extension PCR method, we first fused the mouse DHFR gene, an auxotrophic selection marker, downstream of the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) (see schematic in Fig. 5A). Taking advantage of the modular design of our vector backbone, we then cloned the IRES-DHFR fragment downstream of the Rluc coding sequence, resulting in a bicistronic expression cassette (Rluc-IRES-DHFR) that permits the coexpression of these two genes from a single transcript, the latter through cap-independent translation (Fig. 1E). This vector panel also included an empty control vector without the variable promoter (NP: no promoter).
Schematic representation of stable CHO-DG44 cell line development. (A) Electroporation: CHO-DG44 suspension culture was electroporated with the vector panel. The constructs harbor variable promoters (the CAG promoter is exemplified in this vector map) upstream of the Fluc coding sequence, followed by the CMV-mIE promoter, Rluc gene, IRES sequence and DHFR gene, as well as the Kanamycin resistance gene as the bacterial selection marker. (B) Recovery and auxotrophic selection: Selection was carried out in CD OptiCHO selection media, by sequentially transferring individual CHO-DG44 pools into 6-well plates, T25 flasks and higher volume shake flasks based on their cell division frequency and selection recovery. DHFR based auxotrophic selection took roughly 40 days and cells were occasionally cryopreserved after recovery. (C) Expansion and functional assays: After clonal expansion of stable CHO-DG44 pools in HT deprived media, dual luciferase assay, qRT-PCR based gene expression analysis as well as qPCR based copy number experiments were executed. The schematics are created with BioRender.com.
Development and validation of stably transfected polyclonal CHO-DG44 pools. (A) Schematic representation of IRES-DHFR cassette cloning using the overlapping PCR method. (B) Flow cytometry analysis of electroporated cells after 24 h of nucleofection. pMax-GFP vector was utilized as a transfection efficiency control. GFP positive cell count was detected as 96.9% (on the right). The flow cytometry result on the left indicates untransfected control with 0% GFP positive CHO-DG44 suspension cells. (C) Cell viability and (D) total cell count percentage during DHFR-based auxotrophic selection. In brief, cells were harvested in HT deprived CD OptiCHO selection media and Trypan blue exclusion-based counting was performed every two to three days. Cells were subcultured at 100.000–200.000 cells/ml. Taking into consideration the growth rate of CHO-DG44 pools, the cells were transferred from multiwell plates to orbital shake flasks. (E) The validation of genome integration was performed via conventional non-quantitative PCR using primer sets provided in Table 3. For the CAG promoter, the primer set was altered from P1–P3 pair to P2–P3 that amplifies a shorter fragment with lower GC content (see schematic). Specifically, while P3 reverse primer binds to a region at the 5’ end of the Fluc gene, P2 forward primer targets a region close to the 3’ end of the CAG promoter, that is further verified by Sanger sequencing (data not shown). Note that two different DNA ladders (1 kb DNA ladder—left and 1 kb plus DNA ladder—right) were used in the agarose gel experiments. Expected amplicon and relevant DNA ladder band sizes are highlighted on the agarose gel images. The schematics in (A) and (E) are created with BioRender.com.
The new vector panel was then electroporated into CHO-DG44 suspension cells. Simultaneously, the pMax-GFP vector which encodes the GFP fluorescence protein was used to track nucleofection efficiency. Flow cytometry analysis at 24 h post-transfection revealed that 96.9% of electroporated cells were positive for the GFP signal, indicating a robust transfection efficiency (Fig. 5B). Two days after nucleofection, we applied the auxotrophic selection pressure by culturing the cells in chemically defined commercially available CD OptiCHO media which is naturally devoid of purine precursors (hypoxanthine and thymidine). Every two to three days when sufficient growth was observed, we passaged cells into fresh CD OptiCHO selection media, while transferring them from multiwell cell culture plates to T25 flasks and then orbital shake flasks (see schematic in Fig. 4). During the selection round, we monitored the number of viable and total cell counts at each passage (Fig. 5C,D). As expected, the depletion of untransfected and transiently transfected cells resulted in a rapid and dramatic drop in cell viability and total cell counts. Specifically, we observed that the cell viability decreased from roughly 85–90% to ~ 62% for UBC cells, ~ 43% for SV40 cells, ~ 43% for CMV-mIE cells, ~ 47% for NP cells, ~ 50 for CAG cells, and ~ 47 for CHEF1α cells at day 8 (Fig. 5C). In parallel, total cell counts (per ml) dropped from approximately 0.75–1 × 106 cells to 0.26 × 106 for UBC cells, 0.28 × 106 for SV40 and CMV-mIE cells, 0.3 × 106 for NP cells, 0.36 × 106 for CAG cells, and 0.34 × 106 for CHEF1α cells at day 8 (Fig. 5D). By the end of the second week of selection round, all cell pools except those electroporated with the NP vector developed discernible resistance to selection pressure, as evidenced by an increase in cell viability and total cell count. Meanwhile, the viability of NP cells dropped to a maximum of ~ 13% on day 14. However, on day 17, this value increased to ~ 48%, accompanied by a modest increase in total cell count (Fig. 5C,D). After 3 weeks, cell viability for each CHO-DG44 clone reached about 90–95%, and the cell pools displayed optimal growth characterized by increased viable cell density, indicating that stable transfectant pools had fully recovered from selection pressure (Fig. 5C,D). As predicted, cells electroporated with the pMax-GFP control vector failed to survive the selection pressure and were thus eliminated from the culture (Fig. 5C,D). Next, we sought to confirm that the DHFR-expressing dual luciferase reporter constructs had effectively integrated into the host cell genome. Accordingly, we performed conventional non-quantitative PCR on genomic DNA from all clones using a primer pair that amplifies unique fragments spanning the variable promoter sequences, while sparing the CHO-DG44 genome (see schematic in Fig. 5E). Confirming the genomic integration of each construct, we detected amplicons with expected sizes, particularly, 1404 bp, 561 bp, 809 bp, 225 bp, and 1635 bp for UBC, SV40, CMV-mIE, NP, and CHEF1α, respectively (Fig. 5E). Importantly, no amplification was observed for the CAG fragment, quite possibly due to an inherently high GC content of that promoter (around 65%). To address this issue, we redesigned a new forward primer that was positioned closer to the 3’ end of the CAG promoter. Indeed, the new primer pair supported the amplification of a shorter fragment (190 bp) with lower GC content (roughly 50%), validating the genomic integration of the CAG promoter carrying construct (Fig. 5E).
After developing stable transfectants of polyclonal cell pools with resistance to selection pressure, we performed the dual luciferase assay to quantify the activities of distinct promoters. To this end, we collected Fluc and Rluc bioluminescence signals from three consecutive cell culture passages. In doing so, we intended to test and validate the repeatability and reproducibility of quantitative assessment. In a similar way to the transient expression setting, Fluc to Rluc bioluminescence ratio (Fluc/Rluc) was expressed as a quantitative output of promoter strength. Unlike transient transfection experiments, the CMV-mIE promoter displayed the lowest strength (~ 7) in stable CHO-DG44 pools, whereas the CHEF1α promoter had the highest activity (normalized to 100) that was followed by the SV40 (~ 44), CAG (~ 24), and UBC (~ 12) promoters in Passage 10 (Fig. 6A). Intriguingly, we detected slightly varied promoter activities in Passage 11 and Passage 12; SV40 (~ 41 and ~ 54), CAG (~ 33 and ~ 26), UBC (~ 19 and ~ 14), and CMV-mIE (~ 3 and ~ 5), respectively. Nevertheless, the relative order of promoter strength was the same throughout three passages. It is worth noting that the activity of NP construct was undetectable. Collectively, despite spanning a relatively short period of culture time, we detected similar biological outputs in three consecutive passages which infer that reproducible and consistent transgene expression may be obtained with the current collection of promoters. These findings were further corroborated by mRNA expression of Fluc and Rluc genes in Passage 11 samples, which highly correlated with the bioluminescence data, demonstrating that the luciferase enzyme activities were a direct reflection of transcript levels (Fig. 6B). Finally, we analyzed stable transfectants from the earliest passage for integrated transgene copy number, taking GAPDH gene as a reference. These efforts revealed that both Rluc and DHFR genes exhibited comparable yet slightly divergent copy number values ranging from roughly 1 to 4 copies across different clones which likely complement the heterogeneous nature of polyclonal cell pools (Fig. 6C). According to these findings, we conclude that when successfully implemented to coexpress the DHFR gene as a selection marker, the dual luciferase reporter system is reasonably practical and easily adaptable to assessing regulatory sequences in stably transfected CHO-DG44 clones.
Validation of the vector toolkit in stable transfectants of CHO-DG44 cells. (A) Luciferase activity in stable CHO-DG44 pools electroporated with the select panel of vectors (UBC, SV40, CMV-mIE, CAG, CHEF1α and NP). Luciferase assay was carried out in three consecutive passages (Passage 10 to 12), and each promoter activity was concurrently assessed. Quantification of promoter strengths were carried out by dividing the Fluc signal to the Rluc signal. (B) mRNA expression of Fluc and Rluc genes was verified by qRT-PCR. Much like the luciferase assay results, CHEF1α yielded the highest transcriptional activity in the stable transfection setting, while CMV-mIE displayed the lowest promoter efficacy. (C) Relative DHFR and Rluc copy numbers were calculated according to the internal control gene, GAPDH. Bright Green Master Mix was used for both qRT-PCR assays (B) and transgene copy number analysis by qPCR (C).
