Objective To study the feasibility of using tetracysteine （TC） reporter in gene therapy. Methods Effects of TC reporter and conventional reporter genes encoding green fluorescence protein （GFP） and luciferase （Lu...Objective To study the feasibility of using tetracysteine （TC） reporter in gene therapy. Methods Effects of TC reporter and conventional reporter genes encoding green fluorescence protein （GFP） and luciferase （Luc） on expression and function of the therapeutic gene MGMT^P140K were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry （FCM）. Results The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection. Conclusion TC is a new kind of reporter gene for lentiviral vector in future gene therapy.展开更多
Virus tagged with greenfluorescent protein(GFP)contributes to the visualization and study of the virus in living cells.However,the hepatitis B virus(HBV)particle,which is a compact virion with limited internal space,ca...Virus tagged with greenfluorescent protein(GFP)contributes to the visualization and study of the virus in living cells.However,the hepatitis B virus(HBV)particle,which is a compact virion with limited internal space,cannot be incorporated with GFP tag as a large fragment.It was recently reported that protein genetically inserted with a smaller size tetracysteine(TC)tag could be specially labeled by a biarsenicalfluorescent dye in living cells.In this study,we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV virion.TC tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by mutagenesis.Western blot and enzyme-linked immu-nosorbent assay(ELISA)analysis showed that the TC-tagged core protein,hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)could be expressed in cells transfected with the recombinant HBV vector,which is similar to the cells transfected with wild-type HBV vector.Reverse transcription-polymerase chain reaction(RT-PCR)and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree,but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core proteins.Taken together,the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to befluorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.展开更多
文摘Objective To study the feasibility of using tetracysteine （TC） reporter in gene therapy. Methods Effects of TC reporter and conventional reporter genes encoding green fluorescence protein （GFP） and luciferase （Luc） on expression and function of the therapeutic gene MGMT^P140K were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry （FCM）. Results The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection. Conclusion TC is a new kind of reporter gene for lentiviral vector in future gene therapy.
基金supported in part by the National Natural Science Foundation of China(Grant Nos.30872237 and 30600277)National Key Basic Research Program of China(973 Program,No.2007CB512900)Doctoral Fund of Ministry of Education of China(No.20070487007).
文摘Virus tagged with greenfluorescent protein(GFP)contributes to the visualization and study of the virus in living cells.However,the hepatitis B virus(HBV)particle,which is a compact virion with limited internal space,cannot be incorporated with GFP tag as a large fragment.It was recently reported that protein genetically inserted with a smaller size tetracysteine(TC)tag could be specially labeled by a biarsenicalfluorescent dye in living cells.In this study,we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV virion.TC tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by mutagenesis.Western blot and enzyme-linked immu-nosorbent assay(ELISA)analysis showed that the TC-tagged core protein,hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)could be expressed in cells transfected with the recombinant HBV vector,which is similar to the cells transfected with wild-type HBV vector.Reverse transcription-polymerase chain reaction(RT-PCR)and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree,but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core proteins.Taken together,the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to befluorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.
