Journal: The Journal of Neuroscience
Article Title: Ducky Mouse Phenotype of Epilepsy and Ataxia Is Associated with Mutations in the Cacna2d2 Gene and Decreased Calcium Channel Current in Cerebellar Purkinje Cells
Figure Lengend Snippet: The du mutation is a genomic rearrangement involving the Cacna2d2 gene. a , Two mutant transcripts can be identified by RT-PCR of total brain RNA from du/du mice. Two +/+, two du/du samples, and a negative control (no RNA) are shown per gel. i , Top , Normal size amplification product of exons 1–3 is shown in +/+ and du/du RNA, with reduced levels in the latter. Middle , Amplification between exons 1 and 4 does not produce a product in du/du RNA, suggesting disruption of the Cacna2d2 gene in this region. Bottom , Amplification of the du- specific chimeric transcript of Cacna2d2 exons 1, 2, and 3 and a novel sequence X. ii , Overlapping PCR fragments spanning exons 2–39 of Cacna2d2 can be detected in +/+ and du/du RNA, with lower levels observed in du/du samples. b , Wild-type Cacna2d2 transcript (5.5 kb) is absent from du/du brain by Northern analysis using cerebellar mRNA and full-length Cacna2d2 as a probe. Low levels of two du -specific bands (∼1.5 and 5 kb) are detected. The filter was rehybridized with β-actin as a control for RNA loading. c , PFGE shows duplication of Cacna2d2 exons 2 and 3 and region X in du/du genomic DNA. Southern analysis of Not I-digested genomic DNA separated by PFGE from +/+, +/ du , and du/du mice is shown. Blots were hybridized with Cacna2d2 probes: i , exon 1; ii , exons 2–3; iii , exons 4–39; iv , region X. Sizes are in kilobases. d , A scale representation of the genomic region containing Cacna2d2 ( red ) and region X ( blue ) in +/+ and du/du mice. N , Not I sites. The presence of one or two B2 repeats 5′ to region X is marked by a vertical line . The mutant transcripts 1 and 2 produced from each region in du/du are represented by colored boxes . The Cacna2d2 gene is arranged 5′ to 3′ in +/+. In du/du , exons encoding mutant transcript 1 are shown in a 5′ to 3′ direction, and those encoding mutant transcript 2 are inverted and shown 3′ to 5′. The distance between exon 3 and region X is unknown but is > 12 kb. The scale bar is in kilobases.
Article Snippet: Northern blot analysis of 10 μg of cerebellar mRNA using Duralon UV nylon membrane and full-length Cacna2d2 or human β actin as probes (Stratagene, La Jolla, CA) was performed using the suggested conditions of the manufacturer to optimize the identification of the wild-type 5.5 kb Cacna2d2 transcript.
Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Negative Control, Amplification, Sequencing, Polymerase Chain Reaction, Northern Blot, Produced