The transfection of bone marrow stem cell with Tet-on regulating system and the pilot study of doxycycline depending expression with Tet-on regulating system / 中华骨科杂志

ABSTRACT

Objective To determine the feasibility of using Tet-on regulatory system to transfect primary rabbit bone marrow stem cells, and to control the activities of the target gene. Methods The Tet-on regulatory system were used to transfect bone marrow stem cells by lipofectamine with the increasing concentration of doxycycline (Dox), the activity of the fluorescence caused by the expression of the target gene (the luciferase gene) which transfected to bone marrow stem cells with Tet-on regulatory system were analyzed. Results Plasmids DNA after being digested by enzymes and agarose electrophoreses were analyzed. The DNA segments were cored with the plasmid map. The two DNA segments with the size of 6.02 kb and 1.38 kb could be detected after the plasmid of pTet-on, 5.2 kb DNA segment could be seen after the plasmid of pTRE-luciferase was digested by Hind Ⅲ. With the increasing concentration of Dox, the activity of the fluorescence caused by the expression of the target gene increased gradually. Although the uninfected bone marrow stem cells were induced, the relative-luciferase-unit(RLU) value was almost the same as that of the blanket control. It showed that uninfected bone marrow stem cells were not induced by Dox. The RLU value in infected bone marrow stem cells before induction was slightly high, but similar with the blanket control. This experiment shed light on that infected bone marrow stem cells showed good response to the induction by Dox. Conclusion With the help of Lipofectamine, the cultured bone marrow stem cells can be transfected by Tet-on regulatory system successfully. The expression of the target gene transfected to bone marrow stem cells with Tet-on regulatory system could be regulated by Dox.