[Construction of transgenic RAW264.7 monoclonal cell line by dual-labeling of +8-green fluorescent protein and modified red fluorescent protein phosphatidylserine].
OBJECTIVE
To establish the transgenic RAW264.7 monoclone cell line labelled by green fluorescent protein(GFP) and modified red fluorescent protein(mRFP), for investigating the mechanism of cell membrane fusion and cell biological behavior during osteoclastogenesis.


METHODS
A dual-labeling technique involving GFP and mRFP was applied to RAW264.7 cell line by pVSVG for in situ monitoring of membrane fusion during osteoclastogenesis. The live-cell imaging technology was adopted to consecutively observe the process of osteoclast formation induced by receptor activator of NF-κB ligand(RANKL). Furthermore, tartrate-resistant acid phosphatase staining(TRAP) and 4',6-diamidino-2-phenylindole(DAPI) staining were also used to identify the function and characteristics of RAW264.7 monoclonecell line transfected with phosphatidylserine(PS) and + 8 membrane charge.


RESULTS
The normal morphology of RAW264.7 monoclonecell line transfected with + 8-GFP and PS-mRFP was preserved. The PS and (8+) biosensors co-expressed on the membrane of monocytes. No significant difference of fluorescence density was found. In osteoclasts, (8 + )probes disappeared, while PS expressed in both internal organelles and membrane of osteoclasts. Live-cell imaging observation showed that the multinuclear osteoclasts were generated among monocytes and apocytes. All fusion processes occurred under the condition of cell adherence.


CONCLUSIONS
Successful construction of transgenic RAW264.7 monoclone cell line by GFP and mRFP tags provided a wide field of vision for further investigating the cytoskeleton and organelles of subcellular spatial dimension in osteoclastogenesis.
Department of Periodontology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University & Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China.
L
L
Ren
Matrix Dynamics Group, Faculty of Dentistry, The University of Toronto, Toronto, Canada.
Y Q
YQ
Wang
Matrix Dynamics Group, Faculty of Dentistry, The University of Toronto, Toronto, Canada.
Glogauer
G
Michael
D000818:Animals; D002454:Cell Differentiation; D049452:Green Fluorescent Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; D051379:Mice; D009000:Monocytes; D016328:NF-kappa B; D010010:Osteoclasts; D010718:Phosphatidylserines / Q000032:analysis*; D000067996:RAW 264.7 Cells; D053246:Receptor Activator of Nuclear Factor-kappa B; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; D016328:NF-kappa B; D010718:Phosphatidylserines; D053246:Receptor Activator of Nuclear Factor-kappa B; D000097573:Red Fluorescent Protein