Five g of P1- or P2-GFP constructs were co-transfected with 1 g of pcDNA3 plasmid, containing the neomycin gene (Invitrogen, Cergy-Pontoise, France), using 15 l Lipofectamine 2000 reagent (Invitrogen)

Five g of P1- or P2-GFP constructs were co-transfected with 1 g of pcDNA3 plasmid, containing the neomycin gene (Invitrogen, Cergy-Pontoise, France), using 15 l Lipofectamine 2000 reagent (Invitrogen). alkaline activity, the concomitant appearance of morphological changes (neurites) and PKC (19-36) the increase in the Ms4a6d expression of neuronal markers (nestin, -tubulin III, MAP2) as demonstrated by immunocytochemistry and qPCR. Using these cell-based models, we showed PKC (19-36) that MR expression PKC (19-36) increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively and correlated with MR expression. Finally, while progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by siRNA. Concluding, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases. fragment of the hMR gene, referred to as P1, was amplified from the pGL2-HA plasmid (13) by PCR with GL1 and GL2 primers. This amplicon was thereafter ligated into the sites of pGL3-Enhancer-GFP vector. The fragment was extracted from the H31-P2 plasmid (15) by and digestion and was inserted into the unique site of pGL3-Enhancer-GFP plasmid after filling in all recessive ends with Klenow (Ozyme) treatment and dephosphorylation of pGL3-Enhancer by the Shrimp Alcaline Phosphatase (Promega). Transgenes have been subsequently sequenced to verify their integrity (fragment and the same fragment of hMR gene have been generated. They were obtained from H31-P1 and H31-P2 plasmids (15, 16), in PKC (19-36) which we have inserted the entire coding sequence of the GFP. Transfection Procedures For stable transfection, the 7241d embryonic stem (ES) cell line was used. This cell line has been derived in Cooneys laboratory (Baylor College of Medicine, Houston Texas), from a blastocyst obtained from the breeding of LRH +/? heterozygote mice (17) and was genotyped, phenotyped and characterized as a standard wild type ES cell line (Le Menuet and Cooney, unpublished PKC (19-36) data). The undifferentiated cells 7241d were seeded in 100 mm-Petri dishes one day before transfection. Five g of P1- or P2-GFP constructs were co-transfected with 1 g of pcDNA3 plasmid, containing the neomycin gene (Invitrogen, Cergy-Pontoise, France), using 15 l Lipofectamine 2000 reagent (Invitrogen). Forty-eight hours post-transfection, selection was initiated with 400 g/ml of G418 (PAA, Les Mureaux, France). Neo-resistant clones were picked up after 5 days of G418 selection and propagated using the same medium. Two transfections, for each construct, were carried out, resulting in 11 neo-resistant clones having integrated the P1-GFP construct whereas 4 integrated the P2-GFP construct. In addition, the constructs obtained from H31-P1 and H31-P2 plasmids were stably transfected in undifferentiated ES cells. Cell Culture Mouse ES cells were grown on 0.1% gelatin-coated plates (Sigma-Aldrich, Lyon, France) and on feeder cells (STO Neomycin LIF, kindly provided by Dr Alan Bradley, The Wellcome Trust Sanger Institute, UK) treated with 15 g/ml mitomycin C (Sigma-Aldrich) for 4 hours. Cells were cultured at 37C in a humidified incubator gassed with 5% CO2. Reagents ES medium was composed of DMEM (PAA) containing 15% fetal calf serum (FCS specifically tested for ES culture (AbCys SA, Paris, France), 1X non-essential amino acids (PAA), 2 mM glutamine (PAA), 100 U/ml penicillin (PAA), 100 g/ml streptomycin (PAA), 20 mM Hepes (PAA) and 100 M -mercaptoethanol (Sigma-Aldrich). Embryoid Bodies (EB) medium was similar to ES medium except that it contained 10% FCS without -mercaptoethanol. Neuron medium was similar to EB medium but was supplemented with 5 g/ml insulin (Sigma-Aldrich), 5 g/ml transferrin (Sigma-Aldrich), and 29 nM sodium selenate (Sigma-Aldrich). Differentiation of ES cells into Neuronal-like cells ES cells were cultured on feeder cells for two days after thawing. Subsequently, they were cultured without feeder cells in ES medium with Leukemia Inhibitory Factor (LIF) 1,000 U/ml (AbCys) and differentiation was started after 2 days. To induce EB formation, ES cells were detached, dissociated into single cells with 0.25% trypsin (Invitrogen) and then 1.106 ES cells were plated onto non-adherent bacterial Petri dishes (Greiner Bio-one SAS, Courtaboeuf, France) in ES medium without LIF. EB medium with 1 M all polymerase, and 2 l of the reverse transcription reaction. The PCR cycles were as followed: 95 C for 5 min, specific hybridization temperature for 1 min, 72 C for 1 min, during 1 cycle; 95 C for 45 s, specific hybridization temperature for 45 s, 72 C for 45 s, during 30 cycles; 72 C for 7.