| Objective: To study the possibility of enhanced green fluorescent protein (EGFP) gene as informational gene and select mark in living cell, construct the eukaryotic expression vector pCDNA3.1 (+ ) -EGFP for enhanced green fluorescent protein gene, and transfer the eukaryotic expression vector to Hela cell and rat marrow mesenchymal stem cell,then observe the expression of enhanced green fluorescent protein gene in these two types of cells.Methods: Digest the eukaryotic expression vector pCDNA3.1 ( + ) -EGFP and enhanced green fluorescent protein vector pEGFP-1 with two types of restrictively nucleic acid inscribed enzyme: BamH I and Not I ,obtain linearization pCDNA3.1 ( + ) vector and objective EGFP gene segment, and join them together, consequently gain recombined plasma pCDNA3.1 ( + ) -EGFP, then identify it with BamH I single enzyme digestion and BamH I , Not I double enzyme digestion. Transfer recombined plasma pCDNA3.1 ( + ) -EGFP to cultivated Hell cell and rat marrow mesenchymal stem cell by lipid transferring reagent Lipofect AMINE2000, after selecting them for two weeks (for Hell cell) and four weeks (for rat marrow mesenchymal stem cell) by G418 reagent, obtain positive clone of enhanced green fluorescent protein, observe them with fluorescent microscope after cell creep parcel. Results: Construct the eukaryotic expression vector pCDNA3.1 ( + ) -EGFP for enhanced green fluorescent protein gene successfully, andtransfer it to Hela cell and rat marrow mesenchymal stem cell successfully, observe green fluorescence from positive clone with fluorescent microscope.Conclusion: It is demonstrated that EGFP gene is successfully expressed in Hela cells and rat marrow mesenchymal stem cell as eukaryotic expression vector pCDNA3.1 ( + ) -EGFP, and positive clone can emit green fluorescence, furthermore it can be detected directly in living cells with fluorescent microscope. Therefor EGFP gene is informational gene and select mark in living cell. |
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