title: Expression and cytotoxicity of EGFP-labeled D-amino acid oxidase in HeLa cells.
abstract: To observe the expression of R. gracilis D-amino acid oxidase (DAAO) gene labeled by EGFP in HeLa cells and how it mediates cytotoxicity, DAAO cDNA and EGFP gene were cloned into Eukaryotic expression vector pIRES to construct DAAO gene expression vector pIRES-DAAO. HeLa cells were transfected with pIRES-DAAO in vitro. Expression of EGFP gene in HeLa-D cells was observed under a fluorescent microscope. The efficiency of transfection was analyzed by flow cytometry (FCM). The fluorescent cells were screened by FCM and were named HeLa-D. The activity of HeLa-D cells was detected by MTT colorimetric assay after they were treated with D-Ala at various concentrations. The results indicted that expression of EGFP gene in HeLa-D cells was seen under fluorescent microscope and HeLa-D cells were screened by FCM. Apparently,the prodrug D-Ala killed HeLa-D cells. These results demonstrate that EGFP gene can be regarded as a reporter gene to screen the cells transduced with DAAO quickly, and DAAO/D-Ala suicide gene system may prove helpful in gene therapy of cancer.
authors.affiliation: Department of Cell Biology, Second Military Medical University, Shanghai 200433, China.
authors.forename: Zhi-Ying
authors.initials: ZY
authors.lastname: He
authors.forename: Qian-Li
authors.initials: QL
authors.lastname: Jiang
authors.forename: Yuan-Xiao
authors.initials: YX
authors.lastname: Chen
authors.forename: Li-Min
authors.initials: LM
authors.lastname: Wen
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Yao
authors.forename: Xin-Min
authors.initials: XM
authors.lastname: Wang
authors.forename: Wen-Lin
authors.initials: WL
authors.lastname: Li
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Wang
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Hu
mesh_terms: D000409:Alanine / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003605:D-Amino-Acid Oxidase / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans
chemical_list: D049452:Green Fluorescent Proteins; D003605:D-Amino-Acid Oxidase; D000409:Alanine