A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein

Hu,Dong; Wu,Jing; Xu,Lifa; Zhang,Rongbo; Chen,Liping

doi:10.3892/mmr.2012.988

October 2012 Volume 6 Issue 4

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October 2012 Volume 6 Issue 4

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Article

A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein

Authors:
- Dong Hu
- Jing Wu
- Lifa Xu
- Rongbo Zhang
- Liping Chen
View Affiliations / Copyright

Affiliations: Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China, Institute of Infection and Immunology, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China
Pages: 783-786
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Published online on: July 16, 2012

https://doi.org/10.3892/mmr.2012.988
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Abstract

As the function of autophagy becomes evident in a number of diseases, including cancer and infection, it is crucial to construct macrophage cell lines with stable expression of the microtubule-associated protein light chain 3 (GFP-LC3). In this study, a mouse LC3 open-reading frame was amplified by RT-PCR, and cloned into the pEGFP-C1 plasmid for expression of the GFP-LC3 fusion protein. The recombinant plasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent and stably transfected clones were selected by G418 screening. Autophagic puncta formation was observed by fluorescense microscopy. Additionally, we found that starvation treatment induced a significant increase in the number of autophagosomes, while wortmannin treatment significantly repressed the formation of autophagosomes. This study indicated that the RAW264.7 cell line stably expressing GFP-LC3 is available for use in a GFP-LC3 puncta formation assay, and may contribute to basic investigations of autophagic function or drug screening targeted at autophagy.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein

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Abstract

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