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Updated: Apr 4, 2026
Transfecting RAW264.7 Cells with a Luciferase Reporter Gene
Published on: June 18, 2015
[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3]
Wan Wang1, Qing Zhang1, Runpeng Zhao1
1Department of Medical Immunology and Laboratory, Medical School, Anhui University of Science and Technology, Huainan 232001, China.
Objective:
To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3).
Methods:
A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment.
Results:
The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment.
Conclusion:
The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.
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