Study of intracellular location of adenovirus protein IX with correlative light and electron microscopy based on miniSOG

Objective To establish a method of correlative light and electron microscopy (CLEM) to study the intracellular location of adenovirus protein IX. Methods MiniSOG (mini singlet oxygen generator) is a recently-invented genetically-encoded tag for CLEM. MiniSOG-fused adenovirus IX gene (IXSOG) was cloned by PCR, and inserted into pcDNA3 plasmid to form pTPL-IXSOG, which was used to transfect 293 cells. IXSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG. The transfected cells were fixed in 2.5% glutaraldehyde in situ, stained with diaminobenzidine(DAB) through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location of IXSOG was studied by observing the sections under transmission electron microscope. Results Eukaryotic expression plasmid carrying IXSOG fusion gene was constructed. IXSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections. IXSOG fusion mainly formed punctate aggregations or inclusions in the nucleus. Conclusions The correlative light and electron microscopy method based on miniSOG was successfully established, and it could be used to study the intracellular localization of viral proteins. Key words: MiniSOG; Protein IX; Photooxidation; Correlative light and electron microscopy