1) Gluc vector preparation. If one has a viral vector system which will efficiently transduced the specific cell type that they are interested in, one can subclone the Gluc cDNA into this vector using standard cloning protocols. In general, lentivirus vectors transduce many different cell types with high efficiency. The Gaussia luciferase cDNA, codon optimized for mammalian gene expression4, and a fluorescent protein such as GFP, separated by an internal ribosomal entry site (IRES) elements are cloned into a lentivirus vector under the control of the strong constitutive cytomegalovirus (CMV) promoter to produce LV-Gluc-GFP as described3,10.
2) LV-Gluc-GFP transduction of cells of interest. Plate one million cells in a 2 cm2 well and 24 hrs later, add lentivirus vector so you have a multiplicity of infection (MOI) of 10 - 50 in the presence of 8 μg/ml polybrene. After 5 h, wash the cells with PBS and add fresh culture medium and incubate the cells for 48 hrs. Alternatively, the cells of interest can be transfected by the plasmid expressing Gluc using transfection reagents such as lipofectamine (Invitrogen, Carlsbad, CA), though less efficient and transient. Next, determine the transduction/transfection efficiency by analysis of the CFP expression using fluorescence microscopy. In order to determine the Gluc expression, harvest 5 - 20 μl aliquots of the conditioned medium into a clean white or black 96-well plate, and measure the Gluc activity using a plate luminometer which was set to inject 50 μl 20 μM coelenterazine in DMEM and to acquire photon counts for 10 sec. If lentivirus vector was used to transduce the cells, the gene will integrate within the genome and therefore the cells become stably expressing the reporter, therefore, one may choose to grow and freeze the cells for later use.
3) Measurement of Gluc activity from cells-expressing it. Plate different amount of cells in a different well of a 96-well plate in triplicate. 24 hrs later, aliquot 10 μl of conditioned medium into a black or white plate and measure the Gluc activiy after injecting 50 μl 20 μM coelenterazine and acquire the signal for 10 sec using a luminometer. To monitor cell growth and proliferation, plate one to five thousand cells in a well of 96-well plate in triplicate and the next day, start taking 10 μl aliquots at different time points and measure the Gluc activity as above.
Note: Coelenterazine is known to auto-oxidizes. To stabilize this substrate, it should be diluted in DMEM and stored at R.T in the dark for 30 min before use. PBS with 5M NaCl could be used as an alternative diluent for coelenterazine to give higher light output and more stability of the substrate.
4) In vivo injection of Gluc-expressing cells. Anaesthetize the mice by i.p. injection of ketamine (100 mg/kg) and xylazine (5mg/kg). Inject Gluc-expressing cells or Gluc-expression vectors at the location of interest or systemically (i.v.) and measure Gluc activity in the blood at several time-points before and after injection (see below).
5) Gluc blood monitoring. Blood samples are withdrawn by making a small incision in the tail of mice (no anesthesia required) or by retro-ocular withdrawal (anesthesia required). Typically 5 l blood is withdrawn using a p20 pipette and immediately added to 1 μl 20 mM EDTA and stored at 4ºC until all samples are collected (up to 5 days). Gluc activity is measured using a plate luminometer which is set to inject 100 μl 100 μM coelenterazine (stabilized by incubated for 30 min at R.T.) in DMEM to 5 μl of blood samples and to acquire photon counts for 10 sec. As a background control, blood from mice injected with non-Gluc expressing cells is measured.
6) CCD camera imaging of Gluc signal. Mice are anesthetized as above and i.v. injected via tail-vein with 150 μl coelenterazine (4 mg/kg body weight, around 100 μg/mouse) and photon counts are acquired immediately over 1-5 min using a cooled CCD camera with no illuminationas described4. A light image of the animal is taken in the chamber using dim polychromatic illumination. Following data acquisition, post-processing and visualization is performed using CMIR-Image (a program developed by the Center for Molecular Imaging Research using image display and analysis suite developed in IDL (Research Systems Inc., Boulder, CO) or other programs available from the company from which the CCD camera was purchased. Regions of interest are defined using an automatic intensity contour procedure to identify bioluminescence signals with intensities significantly greater than the background. The mean, standard deviation, and sum of the photon counts in these regions are calculated as a measurement of Gluc activity. For visualization purposes, bioluminescence images are fused with the corresponding white light surface images in a transparent pseudocolor overlay, permitting correlation of areas of bioluminescent activity with anatomy.
Note: Coelenterazine is not soluble in aqueous solution. First, coelenterazine should be dissolved in acidified methanol (add a drop of concentration HCl to 10 ml of methanol) to a concentration of 5 g/l. Immediately before injection, mix 20 μl of coelenterazine with 130 μl PBS and i.v. inject it immediately. A small precipitate/cloudy solution might form during injection which normally does not interfere with imaging. However, if coelenterazine in PBS was incubated at R.T., a larger precipitate will form which might lead to blood-clot. Retro-ocular injections are normally easier to be done and one might choose to inject the substrate this way, rather than tail-vein injection. If this procedure is followed, one might observe some photons around the eye area which can be due to the way the injection was performed and this is normally fine.
In vivo studies. All animal studies should be performed with relevant institutional guidelines and regulations.