[Expression of d-amino acid oxidase gene and green fluorescence protein gene transferred into k562e cells by retroviral vector containing internal ribosome entry site sequence].
The internal ribosome entry site (IRES) sequence was derived from encephalomyocarditis virus. It allows to translate two open reading frames at one mRNA, so two genes conjoined by IRES have the same expression rate. K(DfGC) and K(DfGd) cell lines, stably expressing D-amino acid oxidase (DAAO) gene and green fluorescence protein (GFP) genes, were obtained by transfection of K562e cells with retroviral vector pLDfG containing IRES sequence, DAAO cDNA and GFP gene. Fluorescence positive rate and fluorescence intensity of the two cell lines were measured with flow cytometry. H(2)O(2) production by K(DfGC) and K(DfGd) cells treated with D-alanine was measured by the phenol red oxidation assay. The fluorescence positive rate and fluorescence intensity in K(DfGC) and K(DfGd) cell were 94.64% and 96.31% and 202 units and 174 units per 2 x 10(4) cells, respectively. There was exponential correlation between fluorescence intensity and H(2)O(2) level. The above-mentioned results demonstrate that DAAO gene and GFP gene were simultaneously expressed in K562e cell line by the regulation of IRES sequence, and DAAO level was correlated with fluorescence intensity of GFP.
Zhai
Yong-Ping
YP
Departement of Hematology, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China.
Wang
Jian-Min
JM
Zhang
Yu-Sheng
YS
Zhou
Hong
H
Lu
Shu-Qing
SQ
D001665:Binding Sites / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase