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The study used a humanized, bacteria-originated lux reporter system consisting of six (luxCDABEfrp) genes to express components required for producing bioluminescent signals in human UBC J82, J82-Ras, and SW780 cells without exogenous substrates.
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490 BioTech will be speaking at the SOT 56th Annual Meeting and ToxExpo in Baltimore, Maryland from March 12-16, 2017. This year’s meeting, like its predecessors, is designed to provide
SouthFound Media recently interviewed Dan Close, 490 BioTech’s Chief Science Officer, to discuss our autobioluminescent technology and the newest research we are pursuing. SouthFound specializes in helping Southern startups and
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Abstract: Our group has previously demonstrated the ability to elicit autonomous bioluminescent production from the bacterial luciferase gene cassette (lux) from within the mammalian cellular environment. With an emission spectra
Abstract: Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically
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 you already use to monitor traditional bioluminescent cells, and can generally be interchanged freely with these cells within experimental designs. In short, autobioluminescence is just a simpler, easier way to perform your existing bioluminescent experiments.
Autobioluminescent cells can often be used in place of substrate-dependent cell lines to simplify assay design, reduce costs, and increase the amount 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 any chance for 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, all while providing you with increased data acquisition through 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 grown, 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.
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 cell 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.
No, your existing equipment is all you need. The autobioluminescent signal is simply light at a wavelength of 490 nm, so plate readers and CCD camera-based equipment that can read other bioluminescent signals can usually read the 490 nm signal without any modifications. Please consult your equipment specifications for details to ensure that it is capable of capturing a 490 nm emission signal.
You may not. The autobioluminescent signal is simply light at a wavelength of 490 nm, so plate readers and CCD camera-based equipment can usually read the 490 nm signal without any modifications. If your equipment can be operated without an excitation signal and with an open emission filter, it will likely be compatible with the autobioluminescent signal without modification. Please consult your equipment specifications for details to ensure that it is capable of capturing a 490 nm emission signal and operating without an excitation signal.
The continuously generated signal from autobioluminescent cells allows them to be used in place of many common, more time consuming, and more expensive assay formats. Strong correlations exist between autobioluminescent dynamics and MTT assays or ATP and reactive oxygen species level-dependent cytotoxicological assays. Similarly, for compound detection assays the signal provides an excellent alternative to destructive E-SCREEN assays. However, to ensure strong correlation under your experimental conditions, it is recommended that side-by-side comparisons be performed and appropriate R² values be generated prior to use.
Autobioluminescent cell lines do not require any investigator intervention prior to signal generation. Therefore, they can be treated as required and assayed repeatedly as needed with no special treatment. For long-term assay designs it is recommended that cells be maintained in a temperature/humidity/CO2 controlled environment or returned to an incubator between readings, but no special treatment or handling is otherwise 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 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.
Because autobioluminescent cells have been engineered to self-produce all of the components required for signal generation, the autobioluminescent reaction occurs continuously when the cassette is expressed constitutively. This means that, unlike substrate-dependent luciferase systems such as firefly luciferase, instead of continuously exhausting 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 optimized bacterial 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 mean that the half-life of the autobioluminescent signal is functionally unlimited as long as the cellular population remains healthy.
We have found the autobioluminescent signal to remain stable over multiple passages, however, it is impossible to account for all cellular changes that result from normal growth and maintenance. As such, we recommend maintaining cultures for no more than 10 – 15 passages. This will ensure that the autobioluminescent signal remains stable and will minimize intracellular changes that could affect assay results.
Just as with any transfected cell line, continuous, low level selective pressure will help to ensure the optimized bacterial luciferase gene cassette remains actively expressed from within the cellular genome. While it has been demonstrated that cells often retain their autobioluminescent phenotype even without selective pressure, we do not recommend that antibiotic usage be discontinued during regular growth and maintenance. While doing so can increase cellular growth rates, it can also lead to reductions in autobioluminescent output due to the loss of the cassette from part or all of the population. 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.
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 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.
The minimum number of cells required to observe an autobioluminescent signal is dependent on a number of factors such as your treatment conditions, the sensitivity of the detection equipment, and size of the observed area. We recommend that the minimum cell number be empirically determined 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.
Autobioluminescent output often increases during incubation at room temperature under atmospheric conditions. This is due to changes in the cellular microenvironment that enhance the autobioluminescent reaction. Unfortunately, these same conditions also promote signal output variability. It is recommended that cells not be incubated under atmospheric conditions longer than 15 minutes prior to acquisition, as longer incubations begin to promote increases in signal variability that could deleteriously affect results.
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 optimized autobioluminescent 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% confluence, each well of the 6 well plate, representing each isolated colony, should be harvested and resuspended in 200 µl 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 being performed, each well of the 6 well plate used for transfection should be pooled, resuspended in 200 µl in a single well of a 96 well plate, and used directly for experimental analysis.
The short answer is, none. 490 BioTech’s technology is revolutionary in its ability to continuously and autonomously produce light from human and animal cells. This means that no one yet knows the creative ways it will be used in the future. From personalized medicine that allows doctors to determine how a patients’ cells will respond to potential treatments in real time, to glowing fish that can light up both aquariums and children’s imaginations, 490 BioTech is breaking the barriers of what is considered possible and is ready to help you bring your next big idea to life.