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

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Supporting Material

Posters:

WEBINAR: Using autobioluminescent cells to reduce the cost and complexity of optical imaging (November 2016)

2016-AUTOBIOLUM-WEBINAR

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
  •  Conclusions
FAQs

Basics about autobioluminescence

What is the basic procedure for assaying autobioluminescent output?
What is the minimum acquisition time for assaying autobioluminescent output?
My signal-to-noise ratio is low. Is there a way that I can increase autobioluminescent output in order to improve signal detection?
How well does the autobioluminescent signal correlate with alternative assays such as MTT or MTS assays?

Autobioluminescence advantages over traditional bioluminescence (exp. firefly-luciferase) or reporters (exp. GFP)

How does the signal output compare with reporters like EGFP?
Do you have any data about whether this construct is as bright as other luciferases that do require adding the substrate?
What is autobioluminescence and how is it different from the traditional bioluminescent signal from firefly luciferase?

Autobioluminescence applications in animal models

Are there any intensity differences between subcutaneous and orthotopic tumors development when injected with autobioluminescent cells?
How deep can you image? For example, is it possible to visualize cells in the lungs after IV injection?
Do you have any idea how immunogenic is your luciferase and the substrate which is continuously produced?
Does the system work in formalin fixed tissue section?
Can autobioluminescent cells be visualized within small animal models such as mice?

Autobioluminescence applications in cell lines

Is it possible to make a knock-in cell line to place the autobioluminescent reporter downstream of an endogenous promoter to measure its activity?
How stable is the autobioluminescent signal following multiple passages?
Have you transfected primary cells? And does it require lentivirus packaged vector?
What is the half-life of the synthetic luciferase within autobioluminescent cells?
Is it possible to maintain or assay autobioluminescent cells without G418 or other antibiotic markers?
What are the benefits of using an autobioluminescent cell line vs. a substrate-dependent cell line?
Do autobioluminescent cells require different maintenance protocols than wild type or firefly luciferase-expressing cell lines?
Is there a preferred culture medium for signal acquisition?
What is the minimum number of cells required to observe an autobioluminescent signal?
I am having trouble transfecting the optimized gene cassette into my cell line. How can I improve the efficiency of this process?
Is 12kb too large if I choose to use lentivirus packaging method to knock-in your autobioluminescent cassette?
Is there any special equipment required to assay these cells?

Autobioluminescence Vectors

Are the autobioluminescence vectors ASV-1001 and ASV-1002 viral vectors? And, can they be used in lentiviral expression system?
Will the autobioluminescent vector pEF1αlux (ASV-1002) be suitable for use in mice or mouse cell lines?
Do you have recommendations for which vectors (either CMV or EF1α promoters) are suitable for different cell types? Are there any pros and cons for using one vs the other?
Technical Details

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.

 Autobiolum-cells-fig1

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
In Vivo Imaging
Figure 2. Comparison between substrate-dependent tumor imaging vs. substrate-independent, autonomous tumor imaging techniques highlights the advantages of using ASC's autobioluminescent cell lines such as lower cost, reduced hand-on time, non-invasive tumor tracking in animals and straightforward protocols.

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)?

  1. Inject your subjects with autobioluminescent versions of your tumor cell line
  2. Allow tumors to form using your existing growth protocols
  3. To measure, simply place the subject in the imaging chamber and acquire photon counts
  4. Since there’s no signal activation required, repeat as necessary without additional costs
  5. (Optional) create a standard curve relating cell number to autobioluminescence in vitro to get estimates of total tumor size

 Autobiolum-cells-fig3

 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.

Have Questions?

An Applied StemCell technical expert is happy to help, contact us today!