A Novel Fluorescent Protein Pair for Dual鄄color Two鄄photon Laser Scanning Microscopy*
OBJECTIVE To create far-red fluorescence protein reporter gene mKate2 lentivirus, label human liver cancer cell line HepG2 with lentivirus and explore the feasibility of in vitro fluorescence imaging of labeled tumor cells so as to provide experimental rationales for in vivo fluorescence tumor imaging. METHODS mKate2 gene was amplified from pmKate2-N plasmid. Then the fragment was inserted into the lentivirus expression vector pLenti6.3/V5-DEST. The expression plasmids pLenti6.3-mKate2 and the packaging plasmids were cotransfected into 293T cells. The biological titer of lentivirus was determined. HepG2 cells were infected with mKate2 lentivirus at a MOI (virus multiplicity of infection) of 6 for 96 hours. The infection efficiency was assayed through fluorescence microscope and fluorescent-activated cell scanning (FACS). And 2 × 10(6) mKate2-HepG2 cells were collected for fluorescence imaging through an optical imaging system. And the optimal imaging parameters were determined. RESULTS DNA sequencing analysis confirmed that mKate2 gene sequence was correct and there was no mutation or deletion. The biological titer of produced mKate2 lentivirus was 1.6 × 10(6) TU/ml. At 96 hours after mKate2 lentivirus infection, fluorescence microscope showed that mKate2 was expressed in a large percentage of cells. FACS assay showed that the mKate2 positive rate was 93.8% ± 0.4%. Excitation light 530 ± 15 nm and emission light 710 ± 28 nm were the optimal imaging parameters for mKate2-HepG2 cells. CONCLUSION Lentivirus can mediate efficiently the mKate2 reporter gene labeling of human liver cancer cell line HepG2. The mKate2-labeled HepG2 cells can be detected through in vitro fluorescence imaging. Further tracing studies of in vivo tumor fluorescence imaging are technically feasible.