Enhanced green fluorescent protein labeled Hela cell line.docVIP

 PAGE \* MERGEFORMAT 14 Enhanced green fluorescent protein labeled Hela cell line Of: Yang Jun, Chen Xiaoli, Su Baoshan, Wang Li, Si [Abstract] Objective To construct the enhanced green fluorescent protein (EGFP) labeled Hela cell lines. Methods PCR and DNA sequencing pEGFP C1 eukaryotic expression plasmid, using Qiagen tip 500 for pEGFP C1 eukaryotic expression plasmid DNA extraction and purification, cellfectin transfected Hela cells, G418 selection, limiting dilution monoclonal culture, fluorescence microscopy for the expression of EGFP fluorescence detection, confocal laser fluorescence microscopy detected. Results PCR and DNA sequencing confirmed the correct structure of pEGFP plasmid in the inverted fluorescence microscope under 24h after transfection, some visible green fluorescent Hela cells transfected 72h, about 80% Hela cells were visible green fluorescence. join the selective medium containing G418 selective culture, select a strong fluorescent Hela cells by limiting dilution, screening and cloning continued training, access to stable expression of green fluorescent Hela cell clones, expanding culture and spread to more than 10 passages, this cell line named Hela EGFP. confocal laser microscopy revealed: the Blu-ray ( -395nm) excitation, Hela EGFP cell fluorescence excitation wavelength of green 395nm, the maximum emission peak at 509nm. Conclusion Hela EGFP cell line stably expressing EGFP with the ability for real-time visualization of the mechanism of invasion and metastasis of cervical cancer laid the foundation . [Keywords:] enhanced green fluorescent protein, cervical cancer, Hela cells ABSTRACT: Objective To construct a novel enhanced green fluorescent protein (EGFP) tagged Hela cell subline. Methods EGFP gene fragment was amplified from pEGFP C1 vector and confirmed by PCR and gene sequencing. PEGFP C1 vector DNA was isolated and purified by Qiagen tip 500 for transfection into Hela cells. After Hela cells were transfected with p