Substrate-free Autobioluminescent Cell Lines and Vectors
Applied StemCell has developed a genetically encoded synthetic luciferase bioluminescence system that encodes all of the components required for signal initiation and maintenance. This allows cells to continuously and autonomously produce a bioluminescent signal without the need for additional chemical or fluorescent substrate or stimulation, and without sample destruction.
Decreased costs – no need to purchase luciferin
Increased imaging flexibility – image any time
Easy integration into automated systems – no treatment required for light production
Image the same samples repeatedly without cellular destruction
Reduced hands on time – simply plate and image
Non-invasive in vivo tumor tracking in small animal models - image directly through tissue
WEBINAR: Using autobioluminescent cells to reduce the cost and complexity of optical imaging (November 2016)
Autobioluminescent cells use a genetically encoded synthetic luciferase cassette to continuously produce a bioluminescent signal without the need for extracellular stimulation. By encoding both a luciferase protein, as well as a short synthetic pathway capable of transforming natural intracellular products into luciferin substrates, these cells can self-modulate their bioluminescent production in response to metabolic activity levels, or autonomously enact their bioluminescent phenotype in response to intra- or extracellular events. The use of this self-directed approach to bioluminescent imaging improves upon traditional reporters such as firefly luciferase (luc) by negating the need for light activating chemical substrate addition, which reduces the cost of performance while simultaneously increasing the amount of data that can be obtained per run. This eliminates the need for sample destruction or any investigator interaction, allowing for ultra-simplistic, low-cost bioluminescent screening using existing optical imaging equipment. This webinar will discuss the capabilities and uses of autobioluminescent cells for improving existing bioluminescent imaging workflows and for developing new workflows that leverage the autonomous signal generation phenotype to gather data not available from traditional optical imaging reporter platforms.
Highlights of this webinar:
- An introduction to autobioluminescence
- Autobioluminescent vs. bioluminescent imaging
- Using autobioluminescence for in vitro applications
- Using autobioluminescence for in vivo applications
- Autobioluminescent expression in stem cells
Basics about autobioluminescence
Autobioluminescent cell lines do not require any investigator intervention prior to signal generation. Because of this, they can be treated as required and assayed repeatedly as needed with no special treatment. For assay designs that need to assay for a relatively longer time, I would recommend that the cells be maintained in a temperature/humidity/CO2 controlled environment or returned to an incubator between readings, but otherwise no special treatment or handling is required. For most experimental designs the cells are simply processed as needed, placed into the detector, assayed repeatedly, and then discarded or returned for further growth and/or processing.
The minimum acquisition time required for assaying autobioluminescent output is dependent on a number of factors such as treatment conditions, the number of cells being observed, the sensitivity of the detection equipment being used, and the size of the observed area. We strongly recommend that the acquisition time be empirically determined for each experiment by performing an initial analysis consisting of decreasing acquisition times ranging from 10 minutes to 1 second at each time point you wish to observe.
There are several ways to increase the signal-to-noise ratio of autobioluminescent cells. The most effective method is to reduce the surface area or volume in which the cells are housed (i.e., perform acquisition in a 96 well plate rather than a 24 well plate). If this is not feasible, the number of cells assayed can be increased, opaque plates can be used in place of transparent plates, or a short (< 15 minutes) room temperature pre-incubation can be employed to increase autobioluminescent output.
The continuously generated signal from autobioluminescent cells allows them to be used as a better substitute for many time consuming and cost-inefficient commonly used assays. Strong correlations exist between autobioluminescent dynamics and MTT assays or ATP-dependent cytotoxicity assays. Similarly, for estrogenic compound detection assays the signal provides an excellent alternative to destructive E-SCREEN assays. I would, however, recommend that you start by performing a side-by-side comparison between your current assay and the autobioluminescent assay. If nothing else, this will at least make you appreciate the fact that significantly less labor would be needed using the autobioluminescent assay.
Autobioluminescence advantages over traditional bioluminescence (exp. firefly-luciferase) or reporters (exp. GFP)
Factors such as genetic expression efficiency and host cell metabolic activity level can have a significant impact on the photon output of the synthetic luciferase. However, the most important difference between the synthetic autobioluminescent luciferase and a traditional fluorescent or bioluminescent reporter gene is that it does not rely on any external stimulation to produce its autobioluminescent signal. Because the self-generated luciferin is not oversaturated like it is when externally applied, we suggest that users assume the overall flux of the reaction will be lower than that of an externally stimulated reporter. Nonetheless, the autobioluminescent signal is still easily visualized in most situations, and its continuous nature can leverage the use of increased detection times to overcome circumstances producing significantly lower photonic outputs. Additionally, autobioluminescent signal has an improved signal-to-background ratio compared to fluorescent reporters such as EGFP because there is nearly no autobioluminescent background in cells, animal tissues, and cell culture vessels.
Because the luciferin compound for our autobioluminescent cells is produced internally, it is not over-saturated like those you normally would have to do with other luciferases during the external supplementation. Therefore, we generally observe that these cells produce a lower maximal luminescence relative to cells expressing luciferases that do require adding a substrate. Fortunately, we can usually mitigate this by simply increasing the acquisition time that we use. When we account for the time we save by not having to treat the cells with luciferin and pre-process them before imaging, we still save quite a bit of time even though longer acquisition times are needed.
The only difference between autobioluminescence and traditional bioluminescence is that autobioluminescence is fully self-generated and self-directed by the cell, removing any error or variation resulting from human interaction. This is made possible by engineering the autobioluminescent cells so that they can generate all of the components required to produce a bioluminescent signal internally and without any investigator interaction. This allows them to produce an autobioluminescent signal continuously, or to modulate that signal in response to changes in their environment or genetic expression patterns. Because the autobioluminescent signal is simply light, it can be assayed using the same equipment that is already in use to monitor traditional bioluminescent cells, and the cells can generally be interchanged freely during experiment. In short, autobioluminescence is just a simpler, easier way to perform your existing bioluminescent experiments.
Autobioluminescence applications in animal models
The intensity of the autobioluminescent system will vary based on the host cell’s metabolic activity level, so models that are more metabolically active will produce greater levels of autobioluminescent output. Other important considerations between these models are the size of the tumor and the tumor’s location within the host. Larger tumors will produce more signal than smaller tumors, while equally sized tumors closer to the surface will be easier to be detected due to lower levels of photon absorption by the tissues between the luciferase and the camera.
There are many factors that determine if a luciferase-expressing cell can be visualized within a small animal host. Previous work has demonstrated that autobioluminescent tumors can be visualized following formation within the organs of small animal models, but this does not ensure that they will be appropriate for every application. In general, the self-generation of the luciferin substrate required for autobioluminescent production results in a lower overall flux than substrate-requiring luciferases, so a larger number of cells or a longer signal acquisition time may be required relative to alternative reporter systems for difficult-to-visualize tumor models.
The synthetic luciferase cassette has been successfully expressed within a variety of in vitro cell lines and in vivo small animal models without any observed effects on cellular health or basal metabolic activity. While the immunogenic effects of this system have not been evaluated in all possible models, the available data suggest it has similar immunogenicity to existing fluorescent and bioluminescent reporter genes.
A short answer would be No. Under the current genetic design, the system must be expressed in an intact, metabolically active cell for light to be produced. Because of this, the system would not be functional in formalin fixed tissue sections.
Yes. Autobioluminescent cells can be visualized within small animal models so long as the emission signal is capable of penetrating the tissue between the autobioluminescent cells and the detector. Due to the unique nature of small animal models, it is recommended that preliminary assays be performed in your model system to determine the minimum number of cells and imaging conditions required for reliable detection prior to beginning any new small animal-based research project.
Autobioluminescence applications in cell lines
The autobioluminescent reporter has been engineered so that it can be expressed as a single open reading frame. This allows it to be used similarly to any other reporter gene such that it can be inserted behind a promoter of interest to monitor its activation. When used in this application, it is important to note that some promoters can efficiently drive the expression of short constructs, but produce only limited expression of longer constructs such as the synthetic luciferase cassette. We therefore recommend that this approach only be used with promoters known to be capable of expressing longer constructs to ensure efficient function.
We have found that the autobioluminescent signal remains stable over multiple passages, almost always long enough for us to gather all of the data we need. However, because it is impossible to account for all cellular changes that result from normal growth and maintenance, I would be confident recommending that you can maintain your active cultures for up to 10-15 passages. At this passage level we are confident that the autobioluminescent signal remains stable and this can also minimize the chance that any intracellular changes could affect assay results.
Yes, primary cells have been successfully transfected with the synthetic luciferase gene cassette. The transfection process has been performed using electroporation and transfection by lentivirus. For the transfection of difficult-to-transfect primary cell lines, we highly recommend the use of ASC’s proprietary TARGATTTM technology to assist in integrating multiple copies into the genome.
Because these autobioluminescent cells self-produce all of the components required for signal generation, the autobioluminescent reaction occurs continuously when the synthetic luciferase cassette is expressed constitutively. In other words, unlike substrate-dependent luciferase systems which continuously exhaust a limited supply of externally applied substrate, the autobioluminescent cells will continue to self-synthesize both the luciferase enzyme and its required substrates. This results in continuous autobioluminescent output that is maintained as long as the cell is metabolically and transcriptionally/translationally active. Because the synthetic luciferase cassette is genetically encoded, this phenotype is also passed on to each daughter cell during cell division, allowing populations of cells to be tracked over extended time periods with no external interaction required. These unique attributes make the half-life of the autobioluminescent signal infinite as long as the cellular population remains healthy.
Just as with any transfected cell line, continuous and low level selective pressure will help to ensure that the synthetic luciferase cassette remains actively expressed. That being said, it has been demonstrated that these cells often retain their autobioluminescent phenotype even without selective pressure. In general, I would recommend that selection be maintained during regular growth and maintenance, however, if the potential influence of the antibiotic marker on your specific experimental design is a concern, it can usually be removed during the assay period without significant negative effects.
Autobioluminescent cells can often be used in place of substrate-dependent cell lines to simplify assay design, reduce costs, and increase the amount or time points of data collected in your assays. The autonomous nature of the autobioluminescent signal allows cells to be assayed repeatedly or continuously without destruction, reducing the number of cells that must be prepared for each experiment and reducing sample to sample variability. Because the autobioluminescent signal is completely self-modulated and does not require chemical or photonic stimulation prior to emission, autobioluminescent cells are also more amenable to automated or high throughput assay designs. From a quality control standpoint, the use of autobioluminescent cells removes concerns relating to substrate quality, uptake rates, or application efficiency, and completely removes the chances of unintended substrate/treatment interactions. In general, if a substrate-dependent cell line is used, an autobioluminescent cell line can be substituted to perform the same assay with less cost and less investigator effort, while providing you with increased data acquisition of continuous monitoring.
No. Autobioluminescent cells grow and divide just like wild type or firefly luciferase-expressing cell lines and can be maintained using the protocols you already have in place. In fact, because autobioluminescent cell lines do not require destruction or external stimulation to generate their autobioluminescent signal, they are often easier to maintain. Unlike firefly luciferase-expressing cells, autobioluminescent cells can be assayed repeatedly or continuously, and then returned to the incubator for continued growth. There is no need to prepare individual samples for each time point. The same cell samples can also be assayed repeatedly to provide technical replicates for each time point, increasing your statistical power.
The autobioluminescent signal is strongest when cells are healthy and metabolically active. We therefore recommend that cells be assayed in their preferred culture medium. If possible, phenol red should be omitted from the medium because of its photon absorption properties. However, its effects will be minimal if its use is required and it can be included if necessary.
The minimum number of cells required to observe an autobioluminescent signal really relies on several factors including but not limited to your treatment conditions, the sensitivity of the detection equipment, and size of the observed area. We highly recommend that you determine the minimum cell number for each experiment by performing an initial analysis using decreasing cell populations. We recommend that 5×104 cells/well in a 96 well format be used as a starting point for determining experimentally relevant cell population sizes.
The synthetic luciferase cassette is much larger than a single gene construct such as firefly luciferase, and therefore is often more difficult to transfect. We recommend an electroporation-based transfection protocol due to the large size of the cassette, but other methods have been shown to work as well. In general, the transfection process is detrimental to autobioluminescent output kinetics, and we therefore do not recommend screening at the individual colony level. Following transfection and selection, isolated colonies should be passaged in tandem into paired wells of 24 well and 6 well plates. Upon reaching 85% confluency, each well of the 6 well plate, representing each isolated colony, should be harvested and resuspended in 200 ml in a single well of a 96 well plate. This plate can then be assayed to assess the autobioluminescent output of all clonal lineages simultaneously and the wells from the 24 well plates representing those lineages with the greatest level of autobioluminescent output can be scaled up for further evaluation. If transient transfection is to be performed, each well of the 6 well plate used for transfection should be pooled, resuspended in 200 ml in a single well of a 96 well plate, and used directly for experimental analysis.
The full synthetic luciferase cassette and its associated selection marker have been successfully packaged using the pLenti4/V5 DEST lentiviral expression vector (Life Technologies). The efficiency of packaging was noted to be significantly reduced relative to the packaging of smaller genetic sequences, but the resulting viral particles were capable of successfully transfecting cells.
In almost all cases, your existing equipment is all you need. The autobioluminescent signal is simply lit at a wavelength of 490 nm, so plate readers and CCD camera-based equipment that can read other bioluminescent signals can usually read the autobioluminescent signal without any modifications. For dedicated fluorescent detection equipment, if it can be operated without an excitation signal and with an open emission filter, it will likely be compatible with the autobioluminescent signal without modification.
The pCMVlux vector is not a ready-to-use lentiviral vector. It is a mammalian expression vector pre-assembled for expression of the autobioluminescent gene cassette. As supplied, it can be introduced into cells using any traditional, non-viral transfection procedure. Customers wishing to use viral transfection methods, such as lentiviral expression, can remove the autobioluminescent cassette DNA sequence from the vector and clone it into the packaging vector of their choice using traditional molecular biology approaches.
The pEF1alux expression vector uses a human elongation factor 1 alpha promoter to drive expression of the synthetic luciferase. So long as this promoter is functional in the host cell, the construct will be expressed. While the EF1a promoter has been demonstrated to function in mouse embryonic stem cells, it has not been validated in all mouse cell types. We advise that the customer perform a literature search to determine if there is previous validation of human EF1a promoter function in their cell line of interest prior to purchase.
Studies have shown that, for most cell types, the CMV promoter produces greater autobioluminescent expression than the EF1a promoter. We therefore suggest that the pCMVlux vector be used in most cases. However, in situations where the use of viral-derived promoter sequences is not desired, or for cell types known to silence the CMV promoter, the pEF1alux vector can be substituted with only a minor reduction in signal strength.
Applied StemCell, Inc. (ASC) has developed a genetically encoded synthetic luciferase system based on the bacterial luciferase gene cassette (Figure 1). Unlike traditional bioluminescent systems that encode only the luciferase enzyme, and therefore require the destructive application of a chemical substrate to induce light output, ASC’s synthetic luciferase system encodes all of the components required for signal initiation and maintenance. This allows cells to continuously and autonomously produce a bioluminescent signal without the need for chemical or fluorescent stimulation, and without sample destruction. This revolutionary new approach to optical imaging provides you with increased data acquisition in both tissues and small animal models. By taking advantage of our continuously light producing human cell lines it has finally become possible to break free from the expensive and error-prone introduction of substrate for bio-imaging purposes.
Figure 1. ASC’s synthetic bacterial luciferase is capable of generating a bioluminescent phenotype without sample destruction by utilizing substrates found naturally within the host cell.
ASC's ready-to-use, autonomous bioluminescent cell lines provides users with (figure 2):
- Decreased costs – no need to purchase a separate luciferin
- Increased imaging flexibility – image on your own schedule
- Easy integration into automated systems – no treatment required prior to light production
- Ability to image the same samples repeatedly – no cellular destruction required
- Reduced hands on time – simply plate and image, no additional steps required
- Non-invasive in vivo tumor tracking in small animal models - imaging possible directly through tissue
Our cell lines are ideal for:
- Preclinical drug screening and drug discovery
- Toxicity testing
- Metabolic activity monitoring
- Tumorigenesis and treatment studies
- Estrogenic activity screening
How does autobioluminescent tumor tracking work (figure 3)?
- Inject your subjects with autobioluminescent versions of your tumor cell line
- Allow tumors to form using your existing growth protocols
- To measure, simply place the subject in the imaging chamber and acquire photon counts
- Since there’s no signal activation required, repeat as necessary without additional costs
- (Optional) create a standard curve relating cell number to autobioluminescence in vitro to get estimates of total tumor size
Figure 3. In vivo visualization of live animal subjects as they express bioluminescence from ASC’s autobioluminescent cell lines, allowing fundamental biological processes to be monitored non-invasively. Suitable for preclinical diagnostics, drug discovery, and toxicology research.
Custom Autobioluminescent Cell Line Development
Do you have a specialty or proprietary tissue that can benefit from autobioluminescent expression? Contact us to speak with one of our technology specialists and find out how ASC can modify your existing cell lines to produce data continuously without costly and potentially influential substrate addition or sample destruction. We can quickly modify your samples to express an autobioluminescent phenotype and return them to your laboratory for in-house testing at a fraction of the cost of purchasing and maintaining an existing, substrate-requiring cell line.