[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

Abstract

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.

• Authors: Wan Wang, Qing Zhang, Runpeng Zhao, Xuewei Xu, Yingru Xing, Rongbo Zhang, Jing Wu, Dong Hu
• Journal: Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi) Vol. 31 Issue 9 Pg. 1175-8 (Sep 2015) ISSN: 1007-8738 [Print] China
• PMID: 26359095 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Luminescent Proteins
• Map1lc3b protein, mouse
• Microtubule-Associated Proteins
• red fluorescent protein
• Green Fluorescent Proteins

Topics

• Animals
• Autophagy
• Cells, Cultured
• Green Fluorescent Proteins (genetics)
• Humans
• Luminescent Proteins (genetics)
• Mice
• Microtubule-Associated Proteins (genetics)
• RAW 264.7 Cells