Efficient Expression of Tetracycline- Responsive Gene After Transfection of Dentate Gyrus Neurons In Vitro Jacek Jaworski, 1 Izabela Figiel, 2 Tomasz Proszynski, 1,3 and Leszek Kaczmarek 1 * 1 Nencki Institute, Department of Molecular and Cellular Neurobiology, Warsaw, Poland 2 Nencki Institute, Department of Neurophysiology, Warsaw, Poland 3 Warsaw University, Department of Genetics, Warsaw, Poland 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 trans- fected 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 tetracy- cline analog, doxycycline up to 20%. Application of the Tet-On system resulted in almost 10-fold induction of GFP expression. J. Neurosci. Res. 60:754 –760, 2000. © 2000 Wiley-Liss, Inc. Key words: gene transfer; inducible expression; neuro- nal primary culture The possibility of efficient gene transfer into cells of the nervous system has recently greatly accelerated progress in studies on such diverse phenomena as neuro- genesis, neuronal plasticity, and cell death (Ham et al., 1995; Mayford et al., 1996; Abel et al., 1997; Jordan et al., 1997, Hu and Reichardt, 1999). Unfortunately, the most efficient methods of gene transfer have been proven very sophisticated and time consuming (Latchman, 1996; Silva and Giese, 1998). Recently, an adaptation of the relatively simple calcium phosphate method has been described by Xia et al. (1996) for an efficient and non-toxic transfection of DNA into cortical neurons cultured in vitro. This method has already been used for studies on signaling pathways in apoptosis (Xia et al., 1996; Hetman et al., 1999), as well as gene promoter characterization (Strauss et al., 1997). Many applications of gene transfer would greatly benefit from a method allowing for very tight and well- defined regulation of expression of gene of interest. Un- fortunately, the first generation of technologies permitting control of gene activity suffered from gene expression inefficiency, cell toxicity, as well as unspecific action(s) of the gene inducer applied (for review see: Yarranton, 1992). Recently, several novel approaches for control of gene expression have been described, including Tet-Off and Tet-On systems developed by Bujard’s group (Gossen and Bujard, 1992; Gossen et al., 1995). Both of these systems are based on non-eukaryotic gene promoter ele- ments, derived from E.coli tetracycline operon. Thus, be- cause of the apparent lack of unspecific gene inducer effects, they seem to be a good choice for studying the role of eukaryotic gene expression. Dentate gyrus cultured in vitro provides an interest- ing model to study neuronal function of one of the most widely investigated brain regions (Matson and Kater, 1989). These cultures are derived from postnatal animals and have a mixed, neuronal and glial composition (Figiel and Kaczmarek, 1997a,b). Here we describe a modifica- tion of the calcium phosphate method, originally opti- mized for other neuronal cultures, for efficient gene trans- fection of the cells from the dentate gyrus of the hippocampus. Once having established optimal conditions for the gene transfer, we used the Tet-On variant of Bujard’s system for inducible and very robust expression of transfected gene coding for green fluorescent protein (GFP), placed under the control of tetracycline-regulated promoter. Abbreviations: GFP, green fluorescent protein; DMEM, Dulbecco’s mod- ified Eagle’s medium; PBS, phosphate buffered saline; P hCMV*-1 , tetracy- cline responsive promoter; rtTA, reverse tetracycline transactivator; HBS, HEPES buffered saline; MAP2, microtubule associated protein 2; GFAP, glial fibrillary acidic protein; -gal, E.coli -galactosidase; KRAB, Kru ¨ppel- associated box domain of Kox1 protein. Contract grant sponsor: State Committee for Scientific Research (KBN, Poland); Contract grant number: 4 P05A00715. *Correspondence to: L. Kaczmarek, Nencki Institute, Department of Mo- lecular and Cellular Neurobiology, 02-093 Warsaw, Pasteura 3, Poland. E-mail: leszek@nencki.gov.pl Received 4 January 2000; Revised 24 February 2000; Accepted 28 Febru- ary 2000 Journal of Neuroscience Research 60:754 –760 (2000) © 2000 Wiley-Liss, Inc.