Journal:

Article Title: Novel transcripts of Nox1 are regulated by alternative promoters and expressed under phenotypic modulation of vascular smooth muscle cells

doi: 10.1042/BJ20060300

Figure Lengend Snippet: (A) Schematic diagram of the promoter-luciferase fusion plasmids is shown on the left, where the 5′/3′ ends of the construct relative to the transcription initiation site are indicated. Each construct was transiently transfected into P53LMACO1 (closed bar) or MCE301 cells (opened bar). The β-galactosidase-expression vector was co-transfected as an internal control. The relative luciferase activity was denoted as the ratio to the activity of the pGL3-basic vector. Bars represent means ±SE of at least three experiments.

Article Snippet: The 5′-flanking regions of the corresponding exons for the identified transcripts were isolated from a mouse genomic library (Lambda FIXII Mouse 129/SVJ, Stratagene, La Jolla, CA, U.S.A.), and cloned into a pGL3-basic vector (Promega).

Techniques: Luciferase, Construct, Transfection, Expressing, Plasmid Preparation, Activity Assay

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Targeted disruption of Cacna2d2. A: Strategy for targeted disruption of Canca2d2. Seven 3′-terminal exons of Cacna2d2 replaced by homologous recombination with a neomycin-resistance gene are shown by vertical bars. The recombination caused a decrease in size of the EcoRI restriction fragment, which was revealed by Southern blot analysis with the SalI/EcoRI fragment (hatched box). Restriction sites introduced from the phage lambda cloning site are marked with asterisks. B: Southern blot analysis of wild-type (+/+) and targeted (+/−, −/−) mice. The 8.3-kb EcoRI-band serves as a marker for the targeted C 127 a 127 c 127 n 127 a 127 2 127 d 127 2tm1NCIF allele. C: Northern analysis of Cacna2d2 expression in brains from Cacna2d2-targeted mice: truncated Cacna2d2 mRNA in null-mutants that encodes an aberrant protein devoid of membrane-binding capacity could be detected with a 5′ probe (positions 1742–2862, GenBank Accession No. AF247139), but not with a 3′ cDNA probe that represents the substituted C-terminal part of the α2δ-2 protein (positions 3237–5498). Lane 2 contains twice the amount of RNA to reveal the truncated transcript. D: RT-PCR on total RNA isolated from brain confirms the absence of the targeted 3′ portion of the Cacna2d2 transcript. Lanes 1–3: RT-PCR products generated with a primer set representing positions 1742–2862; Lanes 4–6: RT-PCR products obtained with primers representing positions 3113–4149, GenBank Accession No. AF247139.

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Homologous Recombination, Southern Blot, Clone Assay, Marker, Northern Blot, Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Generated

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Canca2d2-null mice show growth retardation and motor impairment. A: Wild-type mouse (left) next to its Cacna2d2−/− sibling. B: When held vertically by tail, null mutants show hind limb clasping, a common hallmark of neuropathology. C: Spontaneous tonic hind limb extension. D: Null-mutants (bottom lane) show irregularly spaced, outwardly oriented footprints (“ducky” gait) as compared to the wild-type footprints (upper lane).

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques:

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Growth delay and premature death in Cacna2d2−/− mice. A: Differences in body weights between Cacna2d2−/− (n = 7 for weeks 1 to 4; n = 4 for weeks 5 to 8) and control mice (n = 12) are highly significant (P < 0.0001) from week 4 on. B: Kaplan-Meier curves showing highly significant (P < 0.0001) survival differences between Cacna2d2−/− (n = 50) and control mice (n = 214). Censored observations (life spans of sacrificed animals) are shown with vertical bars.

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques:

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Cacna2d2 expression in mouse postnatal cerebellum and embryo. Sagittal section of a P15 normal cerebellum in a bright field (A) and in a dark field (B) after in situ hybridization with a Cacna2d2 probe (white bar corresponds to 0.2 mm). In situ experiment on a 13.5 dpc embryo (C) shows that Cacna2d2 is highly expressed in CNS and sympathetic spinal ganglia (white bar corresponds to 1 mm). Major areas of gene expression are: ol, olfactory lobe; th, thalamus; mb, midbrain; po, pons; mo, medulla oblongata; drg, dorsal ganglia; sc, spinal cord.

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Expressing, In Situ Hybridization, In Situ

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: PCR analysis of CACNA2D2 expression on human heart cDNA panel. Lane 1, adult heart; lane 2, fetal heart; lane 3, aorta; lane 4, apex of the heart; lane 5, atrium, left; lane 6, atrium, right; lane 7, auricle, dextra; lane 8, auricle, sinistra; lane 9, ventricle, left; lane 10, ventricle, right; lane 11, interventricular septum; lane 12, atrioventricular node. The 35-cycle PCR shows significant differences between heart areas in the CACNA2D2 mRNA content. G3PDH transcripts are used as a control.

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Expressing

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Major ECG Parameters in Wild-Type and Cacna2d2 −/− Mice

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Tandem Mass Spectroscopy

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Cacna2d2 null mice show increased seizure susceptibility to the convulsant pentylenetetrazol (PTZ). A: Dose-response curves for induction of clonic seizures demonstrate a greater PTZ sensitivity of Cacna2d2−/− mice compared to wild-type and heterozygous littermates. The curves show logistic fits to the data. The CD50 value obtained by the Spearman-Karber method for the null mutant is 26.6 mg/kg (95% confidence limit (CL): 21.4 to 33.1) compared with 52.0 mg/kg (95%CL: 47.7 to 56.8) and 55.8 mg/kg (95% CL: 49.23 to 63.2), respectively, for Cacna2d2+/− and Cacna2d2+/+ animals. For each genotype, groups of three to six animals were tested at three to four doses. B: Seizure score values with increasing cumulative doses of PTZ. Cacna2d2−/− mice showed significantly greater behavioral seizure score values at the 10 and 20 mg/kg doses compared with Cacna2d2+/+ and Cacna2d2+/− mice (P < 0.008, Kruskal-Wallis analysis of variance); eight animals were tested for each genotype. See “Experimental Procedures” for seizure score scale.

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Endpoint Dilution Assay, Mutagenesis

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Cacna2d2−/− mice have reduced granule cell density and increased apoptosis in cerebellum. H&E-stained sections of cerebellum in null-mutant (A) and control (B) mice at P70 (magnification, ×90) show thinning of granule cell layer, deficiency of Purkinje cells, and presence of Bergmann’s glia (reactive astrocytosis). Arrowheads in (A) indicate picnotic cells or apoptotic bodies. TUNEL (C) and caspase-3 antibody (D) staining show increased amount of apoptotic cells and bodies (arrows) in null mutant. Cerebellar cortex immunostaining with GFAP antibody (brown color) indicates enhanced expression of GFAP, representing glial cell proliferation (gliosis) in Cacna2d2−/− (E) in comparison with wild-type (F) (magnification, ×60).

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Staining, Mutagenesis, TUNEL Assay, Immunostaining, Expressing

Journal:

Article Title: Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

doi:

Figure Lengend Snippet: Comparison of Cacna2d2 Mouse Models

Article Snippet: A replacement-type Cacna2d2 -targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California).

Techniques: Mutagenesis, Sequencing