Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee

Abstract

Background: Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues.

Results: The substrate of luciferase (luciferin) was injected either intraperitonealy (i.p.) or in situ into the muscle or the knee joint. Luminescence resulting from the luciferase-luciferin reaction was measured in vivo with a cooled CCD camera and/or in vitro on tissue lysate. Maximal luminescence of the knee joint and muscle after i.p. (2.5 mg) or local injection of luciferin (50 microg in the knee joint, 100 microg in the muscle) were highly correlated. With the local injection procedure adopted, in vivo and in vitro luminescences measured on the same muscles significantly correlated. Luminescence measurements were reproducible and the signal level was proportional to the amount of plasmid injected. In vivo luciferase activity in the electrotransfered knee joint was detected for two weeks. Intramuscular electrotransfer of 0.3 or 3 microg of plasmid led to stable luciferase expression for 62 days, whereas injecting 30 microg of plasmid resulted in a drop of luminescence three weeks after electrotransfer. These decreases were partially associated with the development of an immune response.

Conclusion: A particular advantage of the i.p. injection of substrate is a widespread distribution at luciferase production sites. We have also highlighted advantages of local injection as a more sensitive detection method with reduced substrate consumption. Besides, this route of injection is relatively free of uncontrolled parameters, such as diffusion to the target organ, crossing of biological barriers and evidencing variations in local enzymatic kinetics, probably related to the reaction medium in the targeted organ. Optical imaging was shown to be a sensitive and relevant technique to quantify variations of luciferase activity in vivo. Further evaluation of the effective amount of luciferase in a given tissue by in vivo optical imaging relies on conditions of the enzymatic reaction and light absorption and presently requires in vitro calibration for each targeted organ.

Figure 1

A Luminescence of tibial cranial muscle and knee joint area after electrotransfer of a luciferase-encoding plasmid and i.p. injection of luciferin. Observation was done 26 minutes after i.p. injection of luciferin (2.5 mg/250 μl) and 7 days after electrotransfer of 3 μg and 60 μg of luciferase encoding plasmid (pC1-luc) into the tibial cranial muscle (on the left) and the knee joint (on the right) respectively. Levels of luminescence are represented in false colours according to a scale from 400 to 1500 (whole scale 65000). 1B and 1C Fluorescence of knee joint after injection of fluorescent albumin. Skin of the leg was removed. Fluorescence was induced with λex = 540 +/- 20 nm and observation was done at λem > 630 nm. Panel B: At these wavelengths, without injecting fluorochrome, it was possible to see bones by transparency. Panel C Just after injection, labelled albumin is concentrated within patella of the knee joint and some diffusion of fluorescence within the joint can be observed. Levels of fluorescence are represented in false colors.

Figure 2

Kinetics of luminescence of tibial cranial muscle and knee joint after electrotransfer of a luciferase encoding-plasmid and either i.p. or local injection of luciferin. 3 and 60 μg of a plasmid encoding luciferase were electrotransfered into the left tibial cranial muscle and right knee joint respectively. Luminescence measurements were done 7 days after electrotransfer. Values are mean +/- SEM of the integrated values of luminescence in region of interest (ROI) of the knee joint or of the tibial cranial muscle. For all experiments, the first measurement was performed 2 minutes after luciferin injection. Panel A: i.p. luciferin injection (2.5 mg/250 μl), n = 5 Panel B: local luciferin injection into the muscle (100 μg/40 μl) and into the knee joint (50 μg/10 μl), n= 5/organ. Luminescence decreased with time in the muscle (t_1/2 = 3.3 minutes), and in the knee joint (t_1/2= 16.5 minutes).

Figure 3

Comparison between the in vivo and in vitro measurements of luciferase activity of tibial cranial muscle after electrotransfer with a luciferase-encoding plasmid. Values are mean +/- SEM (n = 10) of the integrated values of luminescence in ROI of the tibial cranial muscle 8 days after electrotransfer of 0.3, 3 or 30 μg of pC1 luc plasmid as measured in vivo (white columns) and in vitro 3 hours later or more (grey columns) on the same muscles. Individual values are represented by black triangles and the lines link values measured in vivo and in vitro for the same muscles. In vivo background luminescence was subtracted from the luminescence measured. In vitro, background luminescence was negligible. Dose effect: – in vitro **** p < 0.0001 between electrotransfer with 0.3 and 3 μg of plasmid ** p < 0.01 between 3 and 30 μg of plasmid – in vivo $$ p < 0.01 between electrotransfer with 0.3 and 3 μg of plasmid.

Figure 4

Sensitivity of in vivo luminescence measurements and dose effect. Values are mean +/- SEM of the integrated values of luminescence in ROI of the tibial cranial muscle 6 h (n = 6 to 10), 1 day and 8 days (n = 10 to 20) after electrotransfer of 0.3 μg (white columns), 3 μg (clear grey columns) and 30 μg (dark grey columns) of pCMV-luc+ into the tibial cranial muscle. For all points, background luminescence was subtracted. Statistical significance of the differences **** p < 0.0001, ** p < 0.01, * p < 0.05.

Figure 5

Reproducibility of in vivo luminescence measurements on muscles after electrotransfer of a luciferase-encoding plasmid. Values are mean +/- SEM (n = 8 to 10) of the integrated values of luminescence in region of interest (ROI) of the tibial cranial muscle 7 days and 8 days after electrotransfer with 0.3 μg (white columns), 3 μg (clear grey columns) and 30 μg (dark grey columns) of pC1-luc into the tibial cranial muscle. Individual values are represented by black triangles and lines link values for the same muscles at day 7 and 8 after electrotransfer. For each amount of plasmid electrotransferred, luminescence values at day 7 (D7) and 8 (D8) did not differ. For both time of measurement (D7 and D8) a significant dose effect was observed. *** p < 0.001 between 0.3 and 3 μg (for groups D7 or D8). * p < 0.05 between 3 and 30 μg (for group D7 or D8). For all points, background luminescence was subtracted Inset: Linear correlation between values of luminescence measured at day 7 and 8 after electrotransfer. R² = 0.92, p < 0.0001.

Figure 6

In vivo kinetic of luciferase expression in the knee joint and in the tibial cranial muscle and antibody concentration in blood serum after electrotransfer of luciferase-encoding plasmid. Panel A: Mean + SEM (n = 8) of the integrated values of luminescence in region of interest (ROI) of the knee joint at different times after electrotransfer of 60 μg/10 μl of pC1-luc plasmid (black square symbols) or empty plasmid (black diamond symbols). The in vivo luminescence was assayed 2 minutes after luciferin injection into the joint. Panel B: Mean ± SEM (n= 6 to 10) of the integrated values of luminescence in region of interest (ROI) of the tibial cranial muscle at different times after electrotransfer of 0.3 μg (White columns) or 30 μg (grey columns) of pC1 luc plasmid. The in vivo luminescence was assayed 2 minutes after luciferin injection into the electrotransfered muscle. For all point background luminescence was subtracted. Statistical significance for comparisons of luminescence between different times after electrotransfer of 30 μg of plasmid **** p < 0.0001, ** p < 0.01; after electrotransfer of 0.3 μg of plasmid $$ p < 0.01 Inset: Mean + SEM (n = 5) of the concentration of antibodies against luciferase in the blood serum at different times after electrotransfer of 0.3 μg (empty triangles) or 30 μg (black diamon) of pC1 luc plasmid into the muscle.