Luciferase cDNA was amplified by PCR, purified through agarose gel electrophoresis, and subcloned into BamHI and EcoRI sites of pLenti-pgk-puro vector (obtained from Viral Vector Core, UTHSC). The pLenti-Luc-mKate-pgk-puro and pLenti-pgk-puro lentiviral (empty vector for mock transfection) vectors were packaged in 293T cells [13 (link)]. The lentiviral vectors were used to transduce the U87 glioma cell line in the presence of 6 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO). Pools of stably transfected cells (U87Luc and U87Mock) were then selected by growing the cells in 1 μg/ml puromycin. Transduction efficiency was determined by using Nikon Eclipse TE300 fluorescent microscope.
Protocol detail
Lentiviral-Mediated Luciferase Expression
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Corresponding Organization :
Other organizations : University of Tennessee Health Science Center, Fayoum University, Amgen (United States), St. Jude Children's Research Hospital
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Variable analysis
independent variables
- Lentiviral vector type (pLenti-Luc-mKate-pgk-puro and pLenti-pgk-puro)
- Transduction efficiency of U87 glioma cell line
- Polybrene concentration (6 μg/mL)
- Puromycin concentration (1 μg/ml) for selection of stably transfected cells
- Positive control: pLenti-Luc-mKate-pgk-puro lentiviral vector (contains Luciferase cDNA)
- Negative control: pLenti-pgk-puro lentiviral vector (empty vector for mock transfection)
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