Author:

Daniel Saims, Group Leader, In Vitro Operations

Date:

January 19, 2016

Firefly (P. pyralis) luciferase is an enzyme that produces bioluminescence in the presence of D-luciferin. The DNA for the luciferase 2 (luc2) gene, along with a puromycin resistance gene, is incorporated into the genome of the cells using a lentiviral transduction system.

While culturing the cells, puromycin is added to the medium to select only the cells with the inserted luciferase gene. Once the luc-enabled cells are injected into a mouse or rat and D-luciferin is administered intravenously, the light produced can be detected with Molecular Imaging’s IVIS® Spectrum in vivo imaging system (Figure 1). This way, the localization and accumulation of the injected cells can be monitored over time. Most commonly, cancer cells are luc-enabled so that disease progression/regression can be monitored.

Fig. 1: Light from luc-enabled tumor cells detected with the IVIS® Spectrum in vivo imaging system

Figure 1: Light from luciferase-enabled tumor cells detected with the IVIS® Spectrum in vivo imaging system

Along with the luciferase 2 gene, the DNA encoding a fluorescent protein can also be added to the genome of your cells at the same time. After the incorporation of a fluorescent protein gene such as green fluorescent protein (GFP), red fluorescent protein (RFP), or another fluorescent protein of interest, Molecular Imaging can detect the cells using the Attune™ NxT Acoustic Focusing Flow Cytometer (Figure 2) or a fluorescence microscope (Figure 3). After the injection of fluorescently-labeled cells into a mouse or rat, the percentages of these cells in different tissues can be determined over time. Cell cycle analysis, apoptosis analysis, and an analysis of the expression of internal and/or external markers can then be performed on the fluorescently labeled cells.

Fig. 2: GFP detected in the cells using the Attune™ NxT Acoustic Focusing Flow Cytometer.

Figure 2: GFP detected in the cells using the Attune™ NxT Acoustic Focusing Flow Cytometer.

Fig. 3: Brightfield (left) and GFP (right) fluorescence microscopy of luc-enabled cells containing GFP.

Fig. 3: Brightfield (left) and GFP (right) fluorescence microscopy of luc-enabled cells containing GFP.

Since different cell types use different promoters to express genes, a variety of promoters including EF1 alpha, CMV, UbC, and MSCV can be used. Also, for high-efficiency gene delivery, the pseudotype of the virus can be modified to VSV-G or MLV.

If you are interested in luc-enabling your cell lines, contact us today!