Neuronal K + /Cl – co-transporter (KCC2) transgenes lacking neurone restrictive silencer element recapitulate CNS neurone-specific expression and developmental up-regulation of endogenous KCC2 gene Pavel Uvarov,* Priit Pruunsild, To ˜nis Timmusk and Matti S. Airaksinen* *Neuroscience Centre, 00014 University of Helsinki, Helsinki, Finland  Department of Gene Technology, Tallinn University of Technology and National Institute of Chemical Physics and Biophysics, Tallinn, Estonia Abstract The K + /Cl – co-transporter KCC2 maintains the low intracel- lular chloride concentration required for fast synaptic inhibi- tion and is exclusively expressed in neurones of the CNS. Here, we show that the KCC2 gene (alias SLC12a5) has multiple transcription start sites and characterize the activity of 6.8 kb of mouse KCC2 gene regulatory sequence (span- ning 1.4 kb upstream from exon 1 to exon 2) using luciferase reporters. Overexpression of neurone-restrictive silencer factor repressed the reporter activity in vitro, apparently via a neurone restrictive silencer element (NRSE KCC2 ) within intron 1 of the mouse KCC2 gene. In transgenic mice, however, KCC2 reporters with or without deletion of the NRSE KCC2 were expressed exclusively in neurones and predominantly in the CNS with a similar pattern and developmental up- regulation as endogenous KCC2. Moreover, a third trans- gene with just a 1.4-kb KCC2 promoter region lacking the NRSE KCC2 -bearing intron 1 was still expressed predomin- antly in neural tissues. Thus, developmental up-regulation of the KCC2 gene does not require NRSE KCC2 and the 1.4-kb KCC2 promoter is largely sufficient for neurone-specific expression of KCC2. Keywords: chloride co-transporter, c-aminobutyric acid, neurone-restirictive silencer factor, repressor element-1 tran- scription factor, transgenic mice. J. Neurochem. (2005) 95, 1144–1155. The neurone-specific K + /Cl – co-transporter (KCC2) is necessary for fast synaptic inhibition, that is for maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters via GABA type A and glycine receptors (Rivera et al. 1999). KCC2 is an 140-kDa protein with 12 transmembrane domains belonging to the cation chloride co-transporter family (for recent reviews see (Payne et al. 2003; Mercado et al. 2004). KCC2 null mutants die immediately after birth (Hubner et al. 2001) and KCC2 hypomorphic mice develop spontaneous seizures (Woo et al. 2002) and other beha- vioural phenotypes (Tornberg et al. 2005). KCC2 mRNA is expressed only in neurones of the CNS (Payne et al. 1996). Most adult brain neurones express KCC2, although a few subpopulations are negative (Kanaka et al. 2001). During embryonic development, KCC2 mRNA expres- sion follows neuronal maturation; it becomes detectable first in the post-mitotic neurones of brainstem and spinal cord (Li et al. 2002). The mechanisms that mediate the developmental up-regulation of KCC2 are unknown (Ludwig et al. 2003; Titz et al. 2003), although GABA-induced depolarization (Ganguly et al. 2001) and brain-derived neurotrophic factor (BDNF) overexpression (Aguado et al. 2003) have been reported to up-regulate KCC2 expression. In mature animals, Received March 24, 2005; revised manuscript received July 7, 2005; accepted July 13, 2005. Address correspondence and reprint requests to Dr Matti S. Airaksi- nen, Neuroscience Center, Viikinkaari 4, 00014 University of Helsinki, Finland. E-mail: mairaksi@operoni.helsinki.fi Abbreviations used: BDNF, brain-derived neurotrophic factor; DRG, dorsal root ganglion; EST, expressed sequence tag; GAPDH, glyc- eraldehyde-3-phosphate dehydrogenase; KCC2, neuronal K + /Cl – co-transporter; NRSE, neurone-restrictive silencer element; NRSF, neurone-restrictive silencer factor; RACE, rapid amplification of cDNA ends; REST, repressor element-1 transcription factor; RPA, Rnase pro- tection assay; TSS, transcription start site; UTR, untranslated region. Journal of Neurochemistry , 2005, 95, 1144–1155 doi:10.1111/j.1471-4159.2005.03434.x 1144 Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1144–1155