1995 Fiscal Year Final Research Report Summary
A study of pathogenesis, and analysis for clinical and a gene therapy in Duchenne muscular dystrophy.
| Project/Area Number |
06454302
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pediatrics
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
MIIKE Teruhisa Kumamoto University Medical School, Department of Child Development, Professor, 医学部, 教授 (90040617)
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| Co-Investigator(Kenkyū-buntansha) |
SUGINO Shigeto Kumamoto University Hospital, Department of Developmental Pediatrics, assistant, 医学部・附属病院, 助手 (60226446)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Dystrophin / Gene targeting / Transgenic mouse / Promoter / Regulatory element / lacZ / Splicing mechanism / Gene therapy |
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| Research Abstract |
(1) Analysis of dystrophin function :
To determine the functional significane of dystrophin, first we have been trying to produce a muscle type and a brain type dystrophin defective model mouse using gene targeting method, respectively. In spite of our efforts, this project has not been succeeded yet. On the other hand 2 to 3 muscle type dystrophin knock out mice have been reported already, in America and Japan last year. So we started to concentrate to produce a brain type dystrophin knock out mouse.
(2) Mechanism of developmental and tissue specific regulation of muscle and brain type systrophin :
In order to study the mechanism of developmental and tissuespecitic regulation of muscle type dystrophin gene in mice, we generated transgenic mice carrying the 900bp genomic fragment from muscle type dystrophin promoter, fused to the coding region of the bacterial lacZ gene. Ti was found that nineteen mice carried the transgene. Of which six lines showed lacZ expression in the right heart. Th
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e reporter gene expression was detected in presumptive right atria and ventricular myocardium of embryo at 8.5 d.p.c.(dayspost coitus). The results show that the 900bp genomic fragment contain the regulatory element for expression in right heart. Therefore, it is possible to consider that the regulatory regions of skeletal muscle, and also left heat are distinct from the that of right heart within the mouse muscle type dystrophin promoter. As to the brain type, we observed that 2.1kb 5'fragment of the mouse brain type dystrophin gene contains the regulatory element required for its expression in the cerebral cortex, but not in the hippocampus.
(3) Preliminary study for DMD gene therapy :
Our final goal is to convert out of frame of dystrophin gene (DMD) into in frame (BMD) dystrophin gene by controlling RNA splicing (exon skipping) using antisense oligonucleotide. As a preliminary study we are trying to establish two systems to evaluate effect of the gene therapy : 1) Comparing system of dystrophin expression in myotube differentiated from myoblast which was originated in MyoD gene transducted fibroblast from normal and DMD,and 2) System of in-vitro splicing using mini dystrophin gene construct. Less
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