[Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization].
To use green fluorescent protein as a marker to study the localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification, and then cloned into pGEM-Teasy vector. After the double enzyme cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1. Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP). Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed that green fluorescence protein expressed over the whole cell when transfected with vector pEGFP-N1. While after the transfection with pEGFP-ZNF230, the fluorescence located mainly on the nuclei of the cells. We demonstrated that the transfected Cos cell line can express human ZNF230 and mouse znf230 with high efficiency. When transfected with the constructed recombinant pEGFP-ZNF230 vector, the ZNF230 protein localizes mainly on the nucleus.
Xu
Wen-Ming
WM
Department of Medical Genetics, West China Hospital, Sichuan University, and the Key Laboratory of Biotherapy, Ministry of Education, Chengdu 610 041, China. xuwenming@hotmail.com
Zhang
Si-Zhong
SZ
Qiu
Wei-Min
WM
He
Guo-Ping
GP
Liu
Yun-Qiang
YQ
Ma
Yong-Xin
YX
Sun
Yan
Y
D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D050939:Gene Fusion; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C486693:Rnf141 protein, mouse; D014157:Transcription Factors; C440989:ZNF230 protein, human; D049452:Green Fluorescent Proteins