Cell culture models provide substantial information on various cellular responses ranging from exposure to small chemical compounds to genetically mediated target depletion [1
]. In combination with advanced microscopy techniques such as quantitative live-cell imaging they can be directly employed to monitor cellular events and phenotypic changes with spatial and temporal resolution. In vitro cell models are essential tools in basic biomedical research and are applied to identify novel cellular targets and compound candidates in pharmaceutical development [2
]. For imaging-based analysis, expression of fluorescent fusion proteins (FP fusions) is the most commonly used approach to study localization and dynamic changes of proteins in living cells. Commercially available FP-based live-cell assays enable the investigation of processes such as cell proliferation, apoptosis or DNA damage. However, this approach is limited to the visualization of ectopically expressed FP fusions, which may considerably differ from their endogenous counterparts in terms of expression level, activity, localization and protein half-life [4
]. Recently evolved gene editing methods such as the CRISPR/Cas9 or the ZFN/TALEN technology can now be used to generate cell lines expressing fluorescently tagged proteins under their endogenous expression control. While these technologies offer novel opportunities for protein analysis the possibility of functional interference by the attached fluorescent moiety still remains.
Intracellular affinity reagents provide a straightforward approach to overcome the drawbacks of FP fusions as they exclusively visualize and trace the dynamics of endogenous target structures [9
]. Due to their robustness and structural simplicity, fluorescently labelled nanoprobes derived from single-domain antibody fragments of camelids (chromobodies, CBs) can be selected to detect antigens in their native surroundings in living cells [9
]. Since their first description in 2006, numerous CBs have been established and applied to visualize and monitor their target molecules in various cell models and whole organisms [12
While transient expression of CBs is sufficient to visualize the dynamics and relocalization of endogenous proteins on single-cell level, larger screening campaigns require the development of stable cell lines with a homogenous and consistent CB expression [16
]. Although various stable CB cell lines have been reported by us and others, the selection and characterization of cell lines compatible for quantitative live-cell imaging is still very cumbersome. Most importantly, quantitative image analysis often suffers from inconsistent CB expression levels, aggregation and a strong cell-to-cell variance of CB fluorescence intensities (Figure 1
). In this context it has to be considered that in most available cell models CB expression is driven from the strong and constitutively active cytomegalovirus-(CMV) promoter. Although high CB levels are desired in general, elevated transgene expression is sometimes accompanied by misfolding and aggregation [19
]. Additionally, following the conventional workflow of stable cell line generation CB transgenes are usually randomly inserted in the cellular genome (Figure 1
). Notably, this not only bears the risk of unintended genomic manipulation which might affect cellular processes but can also lead to inconsistent CB signal intensities due to the integration of different copy numbers of the CB transgene, chromatin positioning effects [21
] and epigenetic silencing of the promoter upon continuous sub-culturing [22
Here, we explore a combination of previously established methods to improve the generation of stable CB cell lines. Based on comparative analyses we propose to (i) implement our recently developed turnover-accelerated CBs expressed as ubiquitin fusions in order to monitor changes in the antigen concentration and to avoid intracellular CB aggregation, (ii) select a promoter, which is less prone to epigenetic silencing and (iii) insert these optimized CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells using a targeted CRISPR/Cas9 gene editing approach.
2. Material and Methods
2.1. Expression Constructs
All primer sequences, synthesized DNA fragments and plasmids are listed in Table S1
. All expression constructs used in this study are listed in Table S2
The expression constructs encoding for Ub-M-LMN-CB and Ub-R-LMN-CB were generated by amplification the Lamin-NB from the Lamin-CB plasmid [23
] using the following primer set: NB-ubi-for and NB-ubi-rev. The amplified DNA fragment was purified and ligated into PstI and BspEI restriction site of Ub-M-BC1-eGFP and Ub-R-BC1-eGFP (both [24
]), respectively. To generate the Ub-R-BC1-CB containing the EF1-α promoter (referred to as EF1-α_Ub-R-BC1-eGFP), the EF1-α promoter was synthesized as gBlock®
gene fragment (IDT, integrated DNA technologies) and Gibson Assembly [25
] was performed after restriction digest of Ub-R-BC1-eGFP [24
] using the restriction enzymes AseI und NheI. Fragments were assembled using Gibson-Assembly Master Mix (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. The BC1-CB expression construct containing the human β-actin promoter (referred to as h-βact_Ub-R-BC1-eGFP) was generated by amplification of the promoter from pDRIVE-hβ-Actin plasmid ([26
], kindly provided by Hiroyuki Konishi, Aichi Medical University School of Medicine, Japan) using the primer set β-actin-promoter-for and β-actin-promoter-rev and ligation into AseI und NheI digested Ub-R-BC1-eGFP [24
]. To eliminate the PstI restriction site within the hβ-act promoter site-directed mutagenesis was performed utilizing the primer set β-actin-promoter-mutPstI-for and β-actin-promoter-mutPstI-rev using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer’s protocol. For molecular cloning of the AAVS1-CB-donor plasmid (as described in Figure 4A) two DNA fragments were used, which were produced by gene synthesis. At first, AAVS1-CB-donor fragment 1 (gene synthesis, plasmid DNA, IDT) was digested using PciI and MfeI and directly ligated into a PciI and MfeI digested pEGFP-N1backbone (Clontech, Mountain View, CA, USA). Secondly, the resulting plasmid was digested with MfeI and XbaI and completed by AAVS1-CB-donor fragment 2 insertion (gBlock®
gene fragment), which was amplified with the following primer set: AAVS1-CB-donor-fragment-2-for and AAVS1-CB-donor-fragment-2-rev. In a last step the complete cassette was sequence verified using primers Seq-AAVS1-CB-donor-1 - 4 for sequencing. To generate an AAVS1-CB-donor plasmid containing the EF1-α_Ub-R-BC1-CB (resulting plasmid AAVS1_Ub-R-BC1-CB), we used the AseI and XbaI restriction site to integrate the CB cassette including the promoter. For the AAVS1-CB-donor plasmid containing the EF1-α_Ub-R-ACT-CB (AAVS1_Ub-R-ACT-CB) the actin NB was amplified with the primer set NB-ubi-for and NB-ubi-rev and ligated into AAVS1_Ub-R-BC1-CB backbone using PstI and BspEI restriction site. All generated constructs in this study were sequence analysed after cloning.
2.2. Cell Culture, Transfection, Stable Cell Line Generation and Compound Treatment
HeLa Kyoto cells (Cellosaurus no. CVCL_1922) were obtained from S. Narumiya (Kyoto University, Japan), whereas DLD-1 (ATCC®
) and HCT116 (ATCC®
) were obtained from ATCC. All cell lines were tested negative for mycoplasma using the PCR mycoplasma kit Venor GeM Classic (Minerva Biolabs, Berlin, Germany) and the Taq DNA polymerase (Minerva Biolabs). Since this study does not include cell line-specific analysis, all cell lines were used without additional authentication. All cell lines were maintained according to standard protocols. Briefly, growth media containing DMEM (high glucose, pyruvate, ThermoFisher Scientific, Waltham, MA, USA) for HeLa Kyoto and HCT116 cells and RPMI 1640 (ThermoFisher Scientific) for DLD-1 cells supplemented with 10% (v
) foetal bovine serum (FCS, ThermoFisher Scientific) and penicillin-streptomycin (ThermoFisher Scientific) were used for cultivation. Cells were routinely passaged using 0.05% trypsin-EDTA (ThermoFisher Scientific) and were cultivated at 37 °C in a humidified chamber with a 5% CO2
atmosphere. Plasmid DNA was transfected with Lipofectamine 2000 (ThermoFisher Scientific) in HeLa cells, whereas DLD-1 and HCT116 were transfected with TransIT-X2®
(Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. For site-directed integration of the CB into AAVS1 genomic locus, 5 × 105
cells were co-transfected with 2.5 µg of the respective donor plasmid and 2.5 µg plasmid encoding for Cas9 nuclease and gRNA specific for the AAVS1 locus. 24 h post transient transfection cells were subjected to a 48 h selection period using 0.6 µg/mL puromycin dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA). Puromycin-resistant cells were allowed to grow for one week before single clones were isolated. Single clones were analysed regarding the CB expression level by fluorescence microscopy. To verify site-directed integration of the CB-donor plasmid at the AAVS1 locus, genomic DNA of the single clones and the respective parental cell line was isolated using QIAamp DNA mini Kit (QIAGEN, Venlo, the Netherlands). Next, the primer pair genPCR-AAVS1-int-for and genPCR-AAVS1-int-rev [27
] was used for PCR-based genotyping. CB integration into the AAVS1 locus results in an amplicon of ~1400 bps (strategy outlined in Figure 4B). The resulting amplicon was purified and verified via sequence analysis. Compound treatment with FH535 (Sigma-Aldrich) and XAV939 (MedChemExpress) was performed for up to 24 h. For dose-response experiments cells were treated with 1 µM, 10 µM and 50 µM for FH535 and 1 µM, 5 µM and 10 µM for XAV939.
2.3. Fluorescence Imaging, Image Segmentation and Analysis
For fluorescence imaging 5000 cells per well were plated in a black µClear 96-well plate (Greiner Bio-One, Kremsmünster, Austria). For staining of the nuclei in PFA-fixed cells 0.02 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) was used while living cells were continuously incubated with 2 µg/mL Hoechst33258 (Sigma). Images were acquired with an ImageXpress micro XL system (Molecular Devices, San Jose, CA, USA) and analysed by MetaXpress software (64 bit, 22.214.171.1243, Molecular Devices). Fluorescence images comprising a statistically relevant number of cells (>200) were acquired for each condition. For quantitative fluorescence analysis the mean fluorescence of the respective CB expression construct in mCherry or mCherry-CTNNB1 transfected cells was determined. Using the Custom Module Editor (version 126.96.36.199) of the MetaXpress software, we established an image segmentation algorithm that identifies areas of interest based on the parameters of size, shape and fluorescence intensity above local background. To segment the whole cell including the nucleus and the cytosolic compartment, the ectopically antigen mCherry-CTNNB1 or its respective control mCherry was used to generate the corresponding segmentation mask. The average fluorescence intensities were determined for each image followed by subtraction of background fluorescence. From these values the mean fluorescence and standard errors were calculated for three independent biological replicates and student’s t-test was used for statistical analysis.
2.4. Western Blot
DLD-1_AAVS1_Ub-R-BC1-CB cells were seeded in a 10 cm² cell culture dish (Corning) with 3 × 106 cells per dish. After two days the cells were treated with DMSO or 10 µM FH535 for 24 h. For the lysis cells were harvested with a cell scraper and cold PBS and centrifuged at 500× g and 4 °C for 3 min. Per 50 µL pellet 100 µL lysis buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40, 1mM PMSF, 1× protease inhibitor cocktail (Serva, Heidelberg, Germany), 1× phosphatase inhibitor (PhosSTOP, Roche, Basel, Switzerland) 250 µg/µL DNase, 2.5 mM MgCl2) was added. The samples were pipetted 30 times every 10 min for 30 min and centrifuged at 16,000× g for 10 min at 4 °C. The samples were boiled in 2× reducing SDS-sample buffer (60 mM Tris/HCl, pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.02% bromophenol blue) for 15 min at 95 °C. The SDS-PAGE and western blot were performed according to standard procedure. For western blotting the proteins were transferred on nitrocellulose membrane (Amersham, GE Healthcare, Chicago, IL, USA). The blots were scanned on a Typhoon-Trio laser scanner (GE Healthcare). The analysis was done with Image Quant TL Toolbox (GE Healthcare, version 7.0).
For immunoblot detection following antibodies were used: Total-CTNNB1 (mouse monoclonal, BD, #610154, 1:1000), Active-CTNNB1 (mouse monoclonal, 8E7, Millipore, #05-665, 1:500), Tubulin (mouse monoclonal, Sigma-Aldrich, #T9026, 1:2000). For the detection of the primary antibodies fluorescently labelled secondary antibodies (goat-anti-mouse, Alexa Fluor 647, ThermoFisher Scientific, 1:1000) was used.
Considering the importance of cellular imaging in biomedical research and preclinical drug development there is a continuous need for advanced labelling strategies to reliably visualize cellular components in a physiologically meaningful state [3
]. During the last decade, fluorescently labelled nanobodies (chromobodies, CBs) emerged as versatile nanoprobes for cellular imaging of endogenous targets in living cells [9
]. Recently, we demonstrated that quantitative analysis of CB fluorescence can additionally be employed to monitor changes in the concentration of endogenous proteins due to a mechanism termed antigen-mediated CB stabilization (AMCBS) [24
]. To monitor changes in antigen concentration with high precision a strong CB expression has to be combined with a fast CB turnover [24
]. However, for some CBs we previously observed that high expression levels are accompanied with the formation of intracellular aggregates. Here, we showed that expression of CBs as ubiquitin fusions can not only be used to generate turnover-accelerated CBs but also can be applied to reduce the fraction of intracellularly aggregated CBs. Our results are consistent with previous findings reporting an increased solubility and functionality of recombinant proteins expressed as ubiquitin fusions in mammalian cells and bacteria [30
]. While the underlying mechanism is not fully understood, a chaperone-similar function is suggested, where a partially unfolded nascent protein weakly interacts with the nearby, upstream located ubiquitin moiety and thereby transiently precludes unspecific intermolecular interactions [30
To date most stable CB cell lines are still generated by transfection of an expression plasmid followed by antibiotic selection of cells comprising a stable genomic integration of the CB transgene. However, this method is very imprecise since neither the integration site nor the number of integrated copies of the transgene can be adjusted. Additionally, the most widely used CMV promoter is prone to epigenetic silencing [35
]. Accordingly, we noticed heterogeneous and overall weaker CB fluorescence intensities for a multitude of stable CB cell lines upon long-term cultivation. With the EF1-α promoter, we identified a suitable alternative promoter providing strong CB expression and stabilization ratios in the presence of the antigen which is less prone to epigenetic silencing [37
]. However, with the CMV, EF1-α and h-βact promoter we compared only three different promoter types. A more comprehensive analysis of further alternatives including a determination of the methylation status of the promoter regions upon long term cultivation might reveal promoter constructs which are even better suited for a more stable CB expression.
Besides epigenetic modulation, CB expression and thus CB fluorescence is affected by the number of integrated transgenes and chromatin positioning effects [21
]. Here, the CRISPR/Cas9 gene editing technology has been demonstrated as a highly superior approach since it enables the integration of one (heterozygous) or two (homozygous) transgene copies at a predefined genomic site [27
]. In the search of transcriptionally active insertion sites, so-called genomic safe harbour sites (GSH) have been described, which allow robust and stable transgene expression. Most importantly, transgene insertion at GSH does not have adverse effect on the host cell genome. In this context, the adeno-associated virus integration site (AAVS1) on human chromosome 19 was identified as a safe genomic location for integration and high yields of transgene expression [27
]. Based on emerging knowledge regarding CRISPR-targeted genome editing using homology-dependent repair [27
] we used the AAVS1 locus to insert CB-based nanoprobes in human cell models. To validate the versatility and flexibility of this approach, we designed ubiquitin-fused, turnover-accelerated CB expression constructs flanked by AAVS1-specific homology arms (HA-L/R) and introduced actin- (ACT-CB) and CTNNB1-specific CB (BC1-CB) in three human cell model systems.
Although this approach is experimentally straightforward, we faced some problems, which remain to be addressed. Firstly, while the puromycin resistance should only allow the selection of clones that underwent a correct transgene insertion at the AAVS1 locus, we obtained a stable HeLa cell clone with an unspecific integration of the ACT-CB transgene. It can be speculated that due to the increased genomic instability of HeLa cells the CB transgene was inserted randomly [56
]. Notably, for HCT116 and DLD-1 cells our PCR-based genotyping approach revealed only clones with a correct CB insertion at the AAVS1 integration site. Secondly, in this study CB integration was exclusively analysed by PCR-based genotyping which provides no information regarding homo- or heterozygosity of the transgene at the AAVS1 locus or possible off-target integration. This could be analysed by junction PCR using primers binding outside the homology arms or Southern blot analysis [57
Besides targeted genomic integration and expression of a CB transgene from a GSH to visualize an endogenous antigen, gene editing can also be used to directly add a fluorescent protein (FP) to the endogenous protein of interest (PoI) [58
]. However such modifications are restricted either to the N- or the C-terminus of the PoI and from our experience it is not always possible to identify suitable gRNAs to target the intended genomic loci without affecting the integrity of the endogenous protein. Additionally, as repeatedly described, FP tagging can interfere with crucial protein parameters such as turnover, subcellular localization and participation in multi-protein complexes [6
In summary, here we combined previously established methods and conceived a strategy to generate optimized CB expressing cell lines. We illustrate that the expression of CBs as ubiquitin fusion constructs substantially increases solubility and functionality of these intracellular nanoprobes. In addition, by implementing the EF1-α promoter for stable CB expression it can be assumed that unwanted epigenetic silencing during long-term cultivation will be reduced. Lastly, we established a protocol for site-directed integration of turnover-accelerated CBs into the AAVS1 locus by using the CRISPR/Cas9 gene editing technology. By applying this approach, we engineered the AAVS1 locus of three different cell lines (DLD-1, HCT116 and HeLa cells) to stably express turnover-accelerated CBs which are suitable not only to visualize the subcellular localization and dynamics of their respective antigens but also allows the quantification of changes of the endogenous protein levels. Notably, the generated CRISPR donor CB expression vector can be easily modified for integration of different CBs or FPs.
Considering the successful demonstration we are convinced that this approach is a substantial improvement over currently applied strategies to generate stable cell lines comprising intracellularly functional nanoprobes such as CBs. To our knowledge it is the first study describing a targeted insertion of an intrabody into a GSH loci for live-cell imaging. Although not tested yet, it is conceivable that this approach is easy transferable to other live-cell imaging probes such as fluorescently labelled single chain variable fragments (scFvs) [61
]. As those nanoprobes have a high tendency to aggregate, fusion to ubiquitin might be particularly beneficial. However, we have to acknowledge that several aspects such as a comprehensive analysis of other promoters for CB expression, determination of epigenetic modification of the CB transgene upon long term cell cultivation and comparative analysis of additional GSH loci are still lacking. Thus we cannot not judge whether this approach is already optimal or can be further improved. Nevertheless, we expect that our strategy will facilitate the generation of more reliable CB cell models for biomedical research and preclinical compound screening campaigns in the future using advanced cellular imaging.