Molecular biological approaches to neurological disorders including knockout and transgenic mouse models.
Advances of molecular biology have provided a great variety of new approaches to research on human disorders. This article gives an outline of molecular biological approaches to analysis of neurological disorders such as giant cell glioblastoma (GGBM) and amyotrophic lateral sclerosis (ALS), and their respective animal models: p53 knockout mice for GGBM and mutant superoxide dismutase-1 transgenic mice for ALS. Genomic DNA extracted from fresh-frozen tissue is examined by Southern blotting for screening mutations in a certain gene. Polymerase chain reaction (PCR) products of a gene in genomic DNA are examined by single-stranded conformation polymorphism, sequencing and agarose gel electrophoresis for identifying mutations, and for preparing and evaluating DNA probes used in Southern blotting and DNA in situ hybridization (ISH). Total RNA from tissue is examined by northern blotting for quantifying and verifying a certain mRNA. Reverse transcription-PCR products of a certain mRNA in total RNA are examined by sequencing and agarose gel electrophoresis for preparing and evaluating cDNA probes used in northern blotting and mRNA ISH. Tissue total protein is immunoblotted for quantifying and verifying a certain protein, and for evaluating the specificity of antibodies used in western blotting and immunohistochemistry. Immunoprecipitates are immunoblotted for evaluating a profile of protein or other substances. Enzyme-linked immunosorbent assay is used for measuring tissue concentration of protein or other substances, and for determining titers of specific antibodies. By these procedures, chronological analysis of animal models for human diseases contribute to elucidating pathogenic mechanisms and exploiting new therapies. Noticing both the similarity and difference between human and animal disorders will help understand the nature of disease.
Shibata
Noriyuki
N
First Department of Pathology, Tokyo Women's Medical University, Tokyo, Japan. shibatan@research.twmu.ac.jp
Oda
Hideaki
H
Hirano
Asao
A
Kato
Yoichiro
Y
Kawaguchi
Motoko
M
Dal Canto
Mauro C
MC
Uchida
Koji
K
Sawada
Tatsuo
T
Kobayashi
Makio
M
D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics; D000818:Animals; D015139:Blotting, Southern; D002493:Central Nervous System Diseases / Q000235:genetics*; D004195:Disease Models, Animal; D006101:Granuloma, Giant Cell / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008967:Molecular Biology / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction