D-Luciferin

Stability of D-luciferin for bioluminescence to detect gene expression in freely

moving mice for long duration

Nakajima K.1, Hamada K.1, Ito R.1, Yoshida Y.1, Sutherland K.2, Ishikawa M2,3,Ozaki M.4,Shirato H.2,5, Hamada T1,4,61Department of Pharmaceutical Sciences, International University of Health and Welfare,

Ohtawara, Tochigi, 324-8501, Japan.
2Global Center for Biomedical Science and Engineering, Faculty of Medicine, Hokkaido
University, Sapporo, Hokkaido, 060-8012, Japan.
3Faculty of Health Sciences, Hokkaido Universtiy. Sapporo, Hokkaido, 060-8638, Japan.
4Department of Biological Response and Regulation, Faculty of Health Sciences, Hokkaido
University, Sapporo, Hokkaido, 060-0812, Japan
5Department of Proton Beam Therapy, Research Center for Collaborative Projects, Faculty
of Medicine,
6Hakujikai Institute of Gerontology, 5-11-1, Shikahama, Adachi Ward, Tokyo, 123-0864,
Japan.

Key words: circadian rhythm, Period1, in vivo imaging, luciferin
3 Figures

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bio.3917

Abstract

Circadian disturbance of clock gene expression is a risk factor of diseases like obesity, cancer and sleep disorder. For the study of these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate D-luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little is known about the stability of D-luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of luciferin solution over 21 days.

L-luciferin, which is synthesized by racemisation of D-luciferin, had a high concentration after 21 days. In addition, we show that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased comparing with a 1 day solution, although locomotor activity rhythm was not affected. These results show that D-luciferin should be applied to the mouse within at most 7 days to detect the bioluminescence of Per1 gene expression rhythm in vivo.

1.INTRODUCTION
The bioluminescent reporter enzyme firefly luciferase (luc) and its substrate D-luciferin have been used to generate optical imaging signals with high sensitivity in living animals [1]
via the luciferin-luciferase reaction. [2, 3] This phenomenon has been adapted successfully to
whole-body imaging of clock genes using EM-CCD cameras or quantification of clock genes expression by photomultiplier tubes (PMTs).
Detecting the circadian gene expression in the whole body using the bioluminescent reporter enzyme for a long duration enables researchers to study the disturbance of body rhythmicity. Such disturbances are a risk factor of diseases such as obesity, cancer and sleep disorder. [4-6] The bioluminescent reporter enzyme is a good tool to detect the clock gene expression rhythm. However, little is known about the stability of D-luciferin applied to mice over a long duration.
Firefly D-luciferin is a chiral substance. In the firefly, L-luciferin has been reported to synthesize by racemisation of D-luciferin in the body. [7] The chirality of the luciferin substrate is critical to the luciferin–luciferase reaction producing bioluminescence. [8-11] In the present study we explored the stability of D-luciferin and its influence on the monitoring of Per1 gene expression in the liver of freely moving mice. [12]

2.EXPERIMENTAL
2.1Animals
Mice were born and reared in our animal quarters where environmental conditions were controlled at 12 hr light /12 hr dark (LD) cycle with lights on 8:00 – 20:00 {Zeitgeber time (ZT) 0-ZT 12} temperature (23 ± 1 °C) and humidity (50 ± 5%). Animals were given food

and water ad libitum. Light off time (20:00) was designated as ZT 12.
C57BL/6J mice carrying a Per1-promoter driven firefly-luciferase reporter gene (Per1-luc) were used. The Per1-luc reporter was constructed as follows: a 6.7-kb region upstream of the translation-initiation codon of mPer1 was fused to the firefly luciferase coding region. [13]
All animal work was performed in accordance with Guidelines for the Care and Use of Laboratory Animals in International University of Health and Welfare with permission #18014 from the Committee for Animal Experimentation.

2.2HPLC analysis for luciferin quantification.
D-luciferin potassium salt (0.1 mM, Wako Pure Chemical Industries, Japan) was dissolved in distilled water. The solution was kept at room temperature. After 1, 4, 7, 14 and 21 days, the D-luciferin solution was injected into a high-performance liquid chromatography (HPLC) system. For chiral analysis of luciferin, linear gradient elution (15–40% acetonitrile/water with 0.1% TFA, 30 min, 1.0 ml/min) was adopted for separation with a chiral fused-silica column, CHIRAL-CEL OD-RH (4.6 x150 mm; Daicel Chemical Industry, Tokyo, Japan).

D-luciferin and L-luciferin were detected with a fluorescence detector (excitation λ=330 nm,

emission λ=530 nm) as previously reported. [7, 14] The peak time of D-luciferin was around

15 min. The peak of L-luciferin proceeds that of D-luciferin (Fig.1A). These peaks of
D-luciferin and L-luciferin were identified by enzymatic reaction assay from collecting peak samples.[7]

2.3Recording Bioluminescence in freely moving mice
A portable optical detection (POD) device was set in a custom-made recording box (W 400 x D 500 x H 500 mm) equipped with a ventilation system and a monitoring system which measured temperature, humidity and lux.[12, 15] The environmental conditions were controlled

at temperature 23 ± 2℃, humidity 50 ± 5%.

For recording Per1 gene expression in the liver, we developed a tissue contact optical sensor (TCS). [12] The tip area of the optical fiber (3 ~5 mm) was removed from the surface clade. The cut surface of the optical fiber (fiber diameter, 0.50 mm, surface cladding, 0.5 mm thick) was smoothed by our original polishing machine as previous reported. [15] The optical fiber was set in an aluminum sensor box (W 8.0 x D 9.0 x H 2.3 mm) and a plastic scintillator (BC-490, Saint-Gobain crystals) was placed in the space of the sensor box. The four corners of the aluminum sensor box were drilled to make a hole for sutures attaching the sensor to the target organ.
For recording the liver, the TCS was attached using sutures to hook the xiphoid process

and muscle. [15] The optical fiber was passed under the skin and out the neck area. The optical fiber was connected to the POD. At least 7 days after surgery, bioluminescence recording was begun in constant darkness. D-luciferin (after making luciferin solution at 1 and 21 days) was intraperitoneally injected into Per1-luc mice with a controlled flow rate of 10μl/hr based on our previous reports [12, 15] to get enough photon counts.
Photons emitted by the target area of freely moving mice were integrated over 10 sec intervals and averaged for 30 min. These data were plotted and an approximate curve
was made using Excel. The primary peak time of Per1-luc expression was determined as the time of highest intensity of Per1-luc expression around a continuous high-intensity area during each circadian cycle.
Per1-luc transgenic mice were housed in transparent plastic cages (W175 x D245 x H 125 mm) and their locomotor activity rhythm was monitored by an infrared sensor located 30 cm above the surface of the cage using an on-line PC (Chronobiology kit).[12, 15] Daily activity onset was visually estimated from standard double-plot actograms of locomotor activity behavior. The activity onset time was designated as circadian time CT 12.

3.RESULTS AND DISCUSSION
3.1HPLC analysis for luciferin quantification.
Although continuous administration of D-luciferin is needed to examine the Per1 gene expression rhythm, little is known about the stability of D-luciferin. Firefly D-luciferin is a chiral substance and L-luciferin has been reported to be synthesized by racemisation of
D-luciferin in the body.[7] The luciferase is able to recognize the chirality of the luciferin structure, serving as the acyl-CoA synthetize for L-luciferin, whereas D-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin in the body.[7] We therefore examined the stability of D-luciferin over several days. After making the luciferin solution, we kept it at room temperature for 1, 4, 7, 14 and 21 days. These samples were applied to HPLC with a chiral fused-silica column. Figure 1A left shows the
chromatograms of luciferin solution at Day 1. D-luciferin was detected clearly, but there was little L-luciferin. But in the Day 21 sample, the amount of L-luciferin had increased (Fig. 1a right). In Fig. 1b, we show L-luciferin content increase depending the duration; 1, 4, 7, 14 and 21 days sample had the ratio 0.2, 7.3, 11.3. 38.4 and 51.0 %, respectively. L-luciferin contents in 7, 14 and 21 days sample significantly increased (*p<0.05 vs. Day1 sample,
one-way ANOVA followed Dunnett’s test).
3.2The effect of Day 21 sample on Per1 expression and locomotor activity rhythm of Per1-luc mouse
For recording Per1 gene expression in the liver, we used a TCS inserted previously and

intraperitoneally applied D-luciferin into Per1-luc mice using a micro pump.[12, 14, 15] Our previous reports showed that a 20 mg/ml concentration of D-luciferin at a rate of 10μl/hr is enough to detect the bioluminescence of Per1 expression in freely moving condition by EM-CCD camera or TCS. Therefore, in present study we used 20 mg/ml luciferin solution and 10μl/hr injection speed.
Figure 2a and 2c show the expression rhythm of Per1 gene (10 sec intervals recording raw datas) applying the Day 1 sample into the Per1-luc mouse by micro pump in constant darkness. Figure 2B and 2D show photons in Fig.2A and 2B are averaged for 30 min and approximate curve by line. Clear circadian rhythmic expression is detected in the liver. However, using the luciferin solution of the Day 21 sample, the bioluminescent intensity of Per1 gene and the amplitude of Per1 gene expression rhythm are decreased (Fig. 2b, 2d). Representative data of locomotor activity rhythm are shown in Fig. 2e. No luciferin solution had an effect on the locomotor activity rhythm.
The decrease effect of the Day 21 sample on Per1 expression in the liver is summarized in Figure 3a, b and c. There is no difference in the period of Per1 gene expression between Day 1 and Day 21 samples. The peak time of Per1 gene expression also has no significant difference. However, the bioluminescence intensity (photon counts) of Per1 expression is significantly decreased by 70% between Day 1 and Day 21 (p<0.05, Wilcoxon rank sum test). In addition, Figure3d show the amount of total locomotor activity each day while applying the solution had no significant difference.

4.CONCLUSION
Firefly D-luciferin is the specific substrate for the luciferin-luciferase reaction. The bioluminescent reporter enzyme firefly luciferase and its substrate D-luciferin are commonly used to detect the gene expression signals throughout the whole body responding to drugs, environmental cues or diseases. Late responses need to be measured continuously over long periods, especially in diseases that cause disturbances of the circadian rhythms such as obesity, cancer and sleep disorder.
However little is known about the stability of D-luciferin, which has a tendency to change into L-luciferin. L-luciferin, the enantiomer of D-luciferin, behaves as a competitive inhibitor for the bioluminescence reaction of D-luciferin.[9, 10] In the present study, we have shown that a D-luciferin solution becomes a solution including L-luciferin. Seven days after making a
D-luciferin solution, the L-luciferin content is still low (11% in ratio), but at 14 days the ratio has increased to 38% and at 21 days 51%. Increased L-luciferin content in the solution is thought to inhibit the bioluminescence reaction of D-luciferin in the body.
Per1 gene expression in the liver using a Day 21 solution was significantly decreased compared with that of a Day 1 solution. But the Day 21 solution had no effect on circadian regulation of the tissue activity within the body; locomotor activity rhythm was normal. Our

external luciferin application system consists of a micro pump connected to a modified free moving animal with a liquid swivel, balance arm and crisscross mouse harness. A tube was guided into the intraperitoneal cavity through a subcutaneous tunnel from an incision in the
dorsal neck to a ventral abdomen incision.[12, 14, 15] With this system it is convenient to add
additional luciferin solution to measure the gene expression rhythm for long duration. However, it is best that the luciferin solution be applied to the mouse within at most 7 days to detect the bioluminescence of Per1 gene expression rhythm in vivo. Bioluminescence of bmal1 expression in the liver tissue in vitro decreased around 7 days after starting the measurement but increased by refreshment of the culture medium including fresh
D-luciferin[16]. These results can be explained by a failure of the luciferin–luciferase reaction in the cells due to impoverishment of the medium conditions.
It has recently been reported that plants contain self-sustained luminescence.[17] Without
suppling a substrate, autonomous luminescence was produced in tobacco plants from germination to flowering, observing temporal and spatial patterns of luminescence across time scales from seconds to months. However, in rodents there are still no reports of successful auto-bioluminescence. Implanted type application systems can deliver 1μl/hr for 7
days or 0.5μl/hr for 14 days with an osmotic pump (Alzet) [18] or 1μl/hr for several months
with a micro pump[19] to detect the bioluminescence of clock gene expression in freely moving mice. However, in these systems the D-luciferin solution should be applied to the mouse within at most 7 days.

ACKNOWLEDGEMENTS
This research was partially supported by JSPS KAKENHI Grant Number 17H04022 and 18H04724“Resonance Bio”. We are grateful to Dr K. Honma and Dr S. Honma for providing us with the Per1-luc transgenic mice and their technical advice of measuring the bioluminescence.

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Figure1. HPLC analysis for luciferin content
(a)Chromatograms of luciferin solution.
(b)Time course of ratio of L-luciferin to D-luciferin in the solution. Statistical significance as detected by one-way ANOVA followed Dunnett’s test (*P < 0.05 vs. Day 1).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure2. Effect of Day 21 sample on Per1 expression and locomotor activity rhythm of Per1-luc mouse
(a)Per1 gene expression in the liver of freely moving Per1-luc transgenic mouse. D-luciferin was applied soon after making (Day 1 sample).
(b)Per1 gene expression in the liver of freely moving Per1-luc transgenic mouse. D-luciferin was applied 21 days after making (Day 21 sample).
(c, d) Photons emitted by the liver in (a) and (b). Counts were averaged for 30 min. These data were plotted and an approximate curve was made by moving average.
(e) (Left) Locomotor activity rhythm of Per1-luc transgenic mouse in constant darkness. Activity records are expressed in black histograms of activity counts in 5 min bins and double-plotted so that 48h are shown on the x-axis and consecutive days on the y-axis. (Right) Analysis of χ2 periodograms for behavior activity rhythm. The oblique line in the periodogram indicates a significance level of p=0.05.

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Figure3. Decreased effect of Day 21 sample on Per1 expression in the liver
(a)The period of Per1 gene expression in the liver of freely moving Per1-luc mice. Each

column indicates the mean±S.D. (n=3).

(b)The peak time of Per1 gene expression in the liver of freely moving Per1-luc transgenic

mice. Each column indicates the mean±S.D. (n=3).

(c)Luminescence intensity of Per1 gene expression in the liver of freely moving Per1-luc transgenic mice. Each column indicates the mean±S.D. (n=3).
Statistical significance as detected by Wilcoxon rank sum test (*P < 0.05).
(d)Locomotor activity analysis of Per1-luc transgenic mice. Each column indicates the mean ± S.D. (n=3). The mean total activity each day while applying the solution was calculated. Activity in Day 1 sample indicates the ratio of Day 1 samples / (Day 1 +Day 21 samples). Activity in Day 21 sample indicates the ratio of Day 21 samples / (Day 1 + Day 21 samples). The duration of recording of Per1 gene expression is indicated by the two arrows in Fig. 2e.

Stability of D-luciferin for bioluminescence to detect gene expression in freely
moving mice for long duration

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
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