1. Volume 11, Issue 3, Pages 435-443 (March 2005)
Codon-Optimized Gaussia Luciferase cDNA for Mammalian Gene Expression in Culture and in Vivo 
Bakhos A. Tannous, Dong-Eog Kim, Juliet L. Fernandez, Ralph Weissleder, Xandra O. Breakefield 
Molecular Therapy 
Volume 11, Issue 3, Pages (March 2005)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
The marine copepod organism G. princeps. (A) Micrograph of the G. princeps (around 6 mm long from head to telson). Image courtesy of Dr. Steve Haddock, Monterey Bay Aquarium Research Institute (Monterey, CA, USA). (B) Scanning electron microscopic false-colored images of G. princeps. Image courtesy of Tina (Weatherby) Carvalho, University of Hawaii at Manoa (Honolulu, HI, USA).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Comparison of activity of different versions of humanized Gaussia luciferase. 293T cells (0.4 million) in p60 plates were transfected and viable cells, cell-free conditioned medium, or cell lysates were assayed for luciferase activity with 40 μM coelenterazine using a luminometer. Experiments were repeated three times, and similar results were obtained. (A) Signals obtained from viable cells normalized to cell number. Viable cells versus conditioned medium. (B) Signals obtained from cell lysates normalized to micrograms of protein. RLU, relative light units; −SP, signal peptide at N-terminus removed; +KDEL, with KDEL sequence at C-terminus.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Time kinetics of light production from humanized Gaussia (hGLuc), humanized Renilla (hRLuc), and humanized firefly luciferase (hFLuc). 0.3 million 293T cells were transfected with each of the amplicon plasmids. After 48 h, cells were harvested and lysates assayed for luciferase activity with 40 μM coelenterazine (for hGLuc and hRLuc) or 450 μM d-luciferin (for hFLuc) using a luminometer. Experiments were repeated three times and similar data were obtained. (A) Signals were calculated as % RLU of which 100% corresponds to the signal obtained during the first 10 s. (B) Signals obtained from cell lysates were normalized to microgram of cell protein. (C) Emission spectrum of hGLuc and hRLuc. CPS, counts per second.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Effects of increasing coelenterazine concentration on the GLuc, hGLuc, and hRLuc luciferase activity. (A) Combined activity in viable cells and cell-free, conditioned medium. 50,000 infected Gli36 cells (in 100 μl) were added to each well of a 96-well plate. Photon count was acquired for 10 s using the CCD camera after the addition of 100 μl of different concentrations of coelenterazine. (B) Cell lysates were introduced into the luminometer, 100 μl coelenterazine solutions were injected, and bioluminescence was measured. Results are shown from one of three similar experiments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Cell expression comparison of hGLuc, hRLuc, and hFLuc luciferase. Photon count was acquired for 10 s after the addition of 100 μl 40 μM coelenterazine (for hGLuc and hRLuc) or 450 μM beetle d-luciferin (for hFLuc) per well. (A) Different numbers of 293T cells were plated in 96-well plates and transfected with expression plasmids using Lipofectamine reagent (in 100 μl). Signal intensity was obtained from combined viable cells and cell-free conditioned medium using the CCD camera. (B) Intracellular bioluminescence obtained from transfected (as in A) viable cells (after washing off the conditioned medium) measured using a luminometer. (C) Gli36 cells (1 million) in p60 plates were infected with HSV-1 amplicon vectors encoding hGLuc and hRLuc. 48 h postinfection, viable cells were harvested in the conditioned medium and diluted over a range of 20 to 200,000 cells (in 100 μl medium) in 96-well plates and imaged using the CCD camera. This experiment was repeated two times, and similar results were obtained with standard deviation less than 10%.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Bioluminescence obtained from tumor cells expressing hRLuc, hGLuc, and hFLuc in living mice. (A) Gli36 cells (2 million) were infected ex vivo with HSV-1 amplicon vectors encoding hRLuc (left side of animal) and hGLuc (right side) and implanted in nude mice. After 36 h, different amounts of coelenterazine (25, 100, or 200 μg equivalent to 0.96, 3.8, or 7.7 mg/kg body wt) were injected into the animals and the photon count was acquired for 1 min and then for an additional 5 min after substrate injection. (B) Total photon count per minute (photons/min) calculated for tumors in A (n = 4 animals) using the CMIR-Image program. (C) Gli36 cells (2 million) expressing hFLuc (left side of animal) and hGLuc (right side) were implanted into nude mice and imaged 5 days later after injection of 200 μg (7.7 mg/kg body wt) coelenterazine and 4 mg (150 mg/kg body wt) of d-luciferin (n = 4 animals). (D) Photons per minute were calculated as in B for the comparison of hFLuc and hGLuc. (E) One million Gli36 cells expressing hGLuc were implanted in the brains of nude mice and imaged a week later after injection of 100 μg (3.8 mg/kg body wt) of coelenterazine (n = 5 animals). Photon count was acquired for 5 min as in A.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions