Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience

Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here, we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the <i>Gaussia</i> luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The <i>Gaussia</i> luciferase mycosensor assay provides an easy tool to monitor mammalian cell contaminants in a high-throughput fashion

Multiplex blood reporters for simultaneous monitoring of cellular processes

Contains fulltext : 125685.pdf (publisher's version ) (Open Access)Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted Vargula luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted Gaussia luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously

Directed Molecular Evolution Reveals Gaussia Luciferase Variants with Enhanced Light Output Stability

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10–15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics

Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the Gaussia luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially-available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The Gaussia luciferase mycosensor assay provides an easy tool to monitor mammalian cells contaminants in a high-throughput fashion

Multiplex Blood Reporters for Simultaneous Monitoring of Cellular Processes

Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted <i>Vargula</i> luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted <i>Gaussia</i> luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously

The association between erythrocyte-bound apolipoprotein B (ery-apoB) and ABO blood group phenotypes.

<p>The prevalence of the ABO blood group phenotypes per tertile are shown (A). Tertiles are based upon ery-apoB. The first tertile represents the group with the lowest ery-apoB, whereas the third tertile represents the subjects with the highest ery-apoB. The prevalence of ABO blood group phenotypes was significantly different between the three groups (P = 0.002). Ery-apoB levels were almost two-fold increased in subjects with blood group O when compared to subjects with blood group A, B or AB (P-ANOVA <0.001) (B). *P<0.05 when compared to subjects with blood group A, B or AB.</p

Discontinuation of statin therapy did not affect erythrocyte-bound apolipoprotein B (ery-apoB).

<p>Ery-apoB was measured in subjects using statins (N = 54) at baseline and after discontinuing statin therapy for 6 weeks. Individual ery-apoB levels remained fairly stable during the 6 weeks of follow-up since ery-apoB at baseline was strongly correlated to ery-apoB after discontinuing statin therapy for 6 weeks (Spearman r: 0.828; P<0.001).</p

Characteristics of a select group of subjects with measurements of erythrocyte-bound apolipoprotein B (ery-apoB) and ABO blood group phenotype.

<p>Subjects were divided according to ABO blood group phenotype.</p>*<p>Significantly different when compared to blood group O (P<0.05).</p>**<p>Significantly different when compared to blood group A (P<0.05).</p>***<p>Significantly different when compared to blood group A, B or AB (all P<0.05).</p