Efficient expression of tetracycline-responsive gene after transfection of dentate gyrus neurons in vitro.
Gene transfer into neurons both in vivo and in vitro may aid in understanding of gene regulation and function in nerve cells. Especially desirable is ability to control the gene expression. In this study we developed conditions for transfection of hippocampal dentate gyrus neurons in dissociated cultures in vitro by calcium-phosphate method. Furthermore, we describe an effective use of tetracycline responsive gene promoter (Tet-On) system for the controlled and very efficient expression of transfected genes. Under optimal conditions as established in this study, efficiency of transfection of neurons with green fluorescent protein (GFP) driven by constitutive cytomegalovirus (CMV) early promoter reached 2.7%. With tetracycline responsive promoter percentage of GFP-positive neurons raised in the presence of tetracycline analog, doxycycline up to 20%. Application of the Tet-On system resulted in almost 10-fold induction of GFP expression.
Jaworski
J
J
Nencki Institute, Department of Molecular and Cellular Neurobiology, Warsaw, Poland.
Figiel
I
I
Proszynski
T
T
Kaczmarek
L
L
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002130:Calcium Phosphates; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D018891:Dentate Gyrus / Q000166:cytology / Q000502:physiology*; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection / Q000379:methods
D000900:Anti-Bacterial Agents; D002130:Calcium Phosphates; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D013752:Tetracycline; C485829:calcium phosphate, dibasic, anhydrous; D004318:Doxycycline