Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system.
Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.
Huang
C J
CJ
Departments of Neurobiology and Psychiatry, Brain Research Institute, Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, California 90024, USA.
Spinella
F
F
Nazarian
R
R
Lee
M M
MM
Dopp
J M
JM
de Vellis
J
J
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004676:Myelin Basic Protein / Q000235:genetics; D009836:Oligodendroglia / Q000187:drug effects / Q000502:physiology*; D009837:Oligodendroglioma / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D000900:Anti-Bacterial Agents; D008164:Luminescent Proteins; D004676:Myelin Basic Protein; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline