Affymetrix GeneChip Mouse Expression Array MOE430A [MOE430A]

Leukoencephalopathy upon disruption of the chloride channel ClC-2

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ClC-2 is a broadly expressed Cl- channel of the CLC family of Cl- channels and transporters which is abundantly expressed in brain. Here it was proposed to participate in lowering the cytoplasmic Cl- concentration of neurons, a process that establishes an inhibitory response to the neurotransmitters GABA and glycine (Staley et al., 1996). Heterozygous mutations in CLCN2 (the gene encoding ClC-2) were recently reported in a few patients with three clinically distinct forms of epilepsy (Haug et al, 2003). However, the disruption of ClC-2 in mice (ClC-2 KO mouse) did not entail epilepsy (Bösl et al., 2001; Nehrke et al., 2002) but myelin vacuolation in fiber tracts of the central nervous system. We used a gene expression profiling of the ClC-2 KO mouse in brain to identify possible disease mechanism which cause the observed myelin phenotype. As these myelin vacuolation became apparent in the fiber tracts of ClC-2 KO cerebellum at P28 and increased with age, we analysed the cerebellum of ClC-2 KO mice at different postnatal ages, before (P14) and after (P35) the KO cerebellum has been affected by myelin vacuolation.

genotype

Transcription profiling by array of CIC-2 knock out mice

ClC-2 is a broadly expressed Cl- channel of the CLC family of Cl- channels and transporters which is abundantly expressed in brain. Here it was proposed to participate in lowering the cytoplasmic Cl- concentration of neurons, a process that establishes an inhibitory response to the neurotransmitters GABA and glycine (Staley et al., 1996). Heterozygous mutations in CLCN2 (the gene encoding ClC-2) were recently reported in a few patients with three clinically distinct forms of epilepsy (Haug et al, 2003). However, the disruption of ClC-2 in mice (ClC-2 KO mouse) did not entail epilepsy (BÃÂÃÂ¶sl et al., 2001; Nehrke et al., 2002) but myelin vacuolation in fiber tracts of the central nervous system. We used a gene expression profiling of the ClC-2 KO mouse in brain to identify possible disease mechanism which cause the observed myelin phenotype. As these myelin vacuolation became apparent in the fiber tracts of ClC-2 KO cerebellum at P28 and increased with age, we analysed the cerebellum of ClC-2 KO mice at different postnatal ages, before (P14) and after (P35) the KO cerebellum has been affected by myelin vacuolation.

Transcription profiling of chloride channel ClC-2 knock out mice with a Leukoencephalopathy-like phenotype vs.wild type

ClC-2 is a broadly expressed Cl- channel of the CLC family of Cl- channels and transporters which is abundantly expressed in brain. Here it was proposed to participate in lowering the cytoplasmic Cl- concentration of neurons, a process that establishes an inhibitory response to the neurotransmitters GABA and glycine (Staley et al., 1996). Heterozygous mutations in CLCN2 (the gene encoding ClC-2) were recently reported in a few patients with three clinically distinct forms of epilepsy (Haug et al, 2003). However, the disruption of ClC-2 in mice (ClC-2 KO mouse) did not entail epilepsy (Bösl et al., 2001; Nehrke et al., 2002) but myelin vacuolation in fiber tracts of the central nervous system. We used a gene expression profiling of the ClC-2 KO mouse in brain to identify possible disease mechanism which cause the observed myelin phenotype. As these myelin vacuolation became apparent in the fiber tracts of ClC-2 KO cerebellum at P28 and increased with age, we analysed the cerebellum of ClC-2 KO mice at different postnatal ages, before (P14) and after (P35) the KO cerebellum has been affected by myelin vacuolation.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4[]

Title: Affymetrix EukGE-WS2v4 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-47921

Total RNA was prepared from the respective organ by immediate transfer of the frozen pulverized tissue into TRIZOL (1ml Trizol per 70 mg of tissue)(Gibco BRL) and homogenized using a mixer. The homogenized tissue was incubated in TRIZOL at room temperature for 5 min before addition of chloroform (0.2 x vol TRIZOL). The mixture was shaken vigorously for 15 sec and incubated at room temperature for 3 min before centrifugation for 10 min at 4°C with 10000 rpm. The upper phase was transferred carefully into a fresh microtube and isopropanol was added. After vortexing, the tube was incubated for 10 min at room temperature before centrifugation at 14000 rpm, 4°C, for 15 min. The RNA pellets were washed twice with 75% EtOH and dried at room temperature before resuspending in 100 µl DEPC-treated water. The concentration and purity of the RNA was determined by optical density measurement at 260 nm and 280 nm. An aliquot of the RNA (1-2 µg) was analysed by TAE-agarose gel electrophoresis. The total RNA was then further purified using RNeasy columns according to the manufacturer's protocol (Qiagen) and eluted with 35-40 µl DEPC-treated water. Preparation of cDNA from total RNA, 15 µg of purified total RNA were used to prepare cDNA using the Invitrogen double-stranded cDNA kit (# 11917-010). Briefly, the RNA was annealed with 100 pmol HPLC-purified T7(dT)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-TTTTTTTTTTTTTTTTTTTTTTTT) at 70°C for 10 min, quick-chilled on ice and centrifuged briefly. On ice, 4 µl 5x reaction buffer, 2 µl 0.1 M DTT and 1 µl 10 mM dNTPs were added and incubated at 42°C for 2 min to equilibrate the temperature. 2 µl SuperScriptTM II RT (200 U/µl) were added, mixed gently and incubated at 42°C for 1 hour. The reaction was placed on ice, spun briefly and the second strand synthesis ingredients were added (91 µl DEPC-H2O, 30 µl 5X Second Strand Reaction Buffer, 3 µl 10 mM dNTP mix, 1 µl E.coli DNA Ligase (10 U/µl), 4 µl E.coli DNA Polymerase I (10 U/µl), 1 µl E.coli RNase H (2 U/µl)) before incubation at 16°C for 2 hrs. 2 µl T4 DNA Polymerase (5 U/µl) were added and the reaction was incubated for another 5 min at 16°C. The reaction was stopped by addition of 10 µl 0.5 M EDTA and phenol-chloroform extracted. The upper phase (approximately 140-150 µl was transferred to a fresh microtube and the cDNA was precipitated with 0.5 x vol 7.5 M NH4OAc and 2.5 x vol ice-cold absolute EtOH by vortexing and immediate centrifugation for 20 min at room temperature. The pellet was washed twice with ice-cold 70% EtOH and dried at 37°C for 10 min before resuspending in 12 µl DEPC-treated water.

P-MEXP-47922

Preparation of Biotin-labelled cRNA from cDNA and fragmentation of the cDNA for hybridization Biotin-labelled cRNA was produced with the BioArray HighYield RNA Transcript Labeling Kit (ENZO) following exactly the manufacturer's instructions. 1/2 of the cDNA (6 µl) was used as starting material and the reaction was incubated in a water bath at 37°C for 5 hours, gently mixing the contents of the tube every 30 min during the incubation. The labelled cRNA was purified with RNeasy Mini colums (QIAGEN) and eluted with 40 µl DEPC-treated water. 1 µg of purified and non-purified cRNA was analysed on a 1% TAE-agarose gel. The measured amount of labelled cRNA (by OD 260) was adjusted to the real amount of labelled cRNA by substracting the amount of starting total RNA (15 µg) from the measured total amount of cRNA. 15 µg of labelled cRNA (adjusted amount) per standard Affymetrix microarray were fragmented in a total volume of 30 µl by incubating with 1 x fragmentation buffer (provided as a 5 x stock: 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) at 94°C for 30 min. 1 µg of fragmented cRNA (2 µl) were analysed by TAE-agarose gel electrophoresis.

P-MEXP-47929

Mice are housed under normal conditions in the animal facility of the UKE Hamburg, with the light in the room being on from 7 a.m. to 7 p.m.. They are fed with standard food from the company Ssniff (R/M Haltung)ad libitum. After killing the mice by cervical dislocation, the organs of interest were taken out, put in liquid nitrogen, and pulverized. The next step was the RNA extraction. 

Transcription profiling by array of CIC-2 knock out mice

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