CELL LINES
Contributor Information
- Name Jose Luis Pedraz ; Laura Saenz del Burgo ; JesÄ?s Ciriza
- Institute University of the Basque Country (EHU)
Tool Details
- Tool name: Luciferase-expressing D1-MSC cell line
- Tool type: Cell Lines
- Parental cell line: D1-MSC
- Organism: Mouse
- Tissue: Bone Marrow
- Growth properties: Adherent cells
- Model: Stem Cells
- Conditional: No
- Description: Mesenchymal stem cells (MSCs) are multipotent stem cells found in bone marrow that are important for making and repairing skeletal tissues, such as cartilage, bone and the fat found in bone marrow. D1-MSC's are a cell line of murine MSCs. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. Luciferases are widely used in biotechnology, for microscopy and as reporter genes, for many of the same applications as fluorescent proteins. However, unlike fluorescent proteins, luciferases do not require an external light source, but do require addition of luciferin, the consumable substrate. The detection of luciferase can be used to track the location of MSCs by non invasive image techniques.
- Research area: Developmental biology; Stem cell biology
- Production details: pSIN-EF2-Luc-Pur was transfected into One Shot1 Stbl3TM Chemically Competent E. coli, extracted with QIAprep1 Spin Maxiprep Kit (Qiagen) and quantified with Nanodrop ND-1000 Spectrophotometer. 2.5 x106 293T cells were cultured with DMEM, 10% FCS and 1% P/S overnight and further transfected with 9mg of pSIN-EF2-Luc-Pur 3mg of pMD2.G (Addgene plasmid 12259), 6mg of psPAX2 (Addgene plasmid 12260) and 45mL of lipofectamine1 2000 in OptiMEM medium following providerÄË?Â?Âs recommendations. After 3 days at 37 C, supernatants from transfected 293 T cells were collected, spun up for 10 min at 2000 g filtered with a 0.45mm PVDF low retention Millex1 filter (EDM Millipore), and stored in liquid nitrogen on 1 mL aliquots until further use. D1-MSCs cells were plated in 6 well-plates at a density of 5 x104 cells/well. A final volume of 2 mL with 1800mL of stored supernatant containing viruses codifying Luc-Pur and 4mg/mL of polibrene were added next day to D1-MSCs. Cells were incubated for 48 h and supernatant was replaced with new maintenance medium containing puromycin 12.5mg/mL. D1-MSCs were cultured and expanded with medium and 12.5mg/mL puromycin for 1 month to ensure all cells were infected. For assessing lack of virus production by infected cells, supernatant from infected cells cultures was transferred to 105 virus-free D1-MSCs and incubated for 3 days. ÄË?Â?ÂRe-infectedÄË?Â? cells were collected and their genomic DNA extracted with QIAamp DNA Mini Kit (Qiagen). Insertion of Luc cDNA sequence into the genome was assessed by PCR following the same parameters than for Luc cloning.
- For Research Use Only
Target Details
- Target: Luciferase
Application Details
Handling
- Format: Frozen
- Growth medium: DMEM supplemented with 10% FCS and P/S
- Shipping conditions: Dry ice
Documentation
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