2005 Fiscal Year Final Research Report Summary
Identification of causative genes for congenital malformation by ENU mutagenesis using medaka fish
| Project/Area Number |
16591011
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Pediatrics
|
|---|
| Research Institution | Niigata University |
|---|
Principal Investigator |
SATOKATA Ichiro Niigata University, Institute of Medicine and Dentistry, Professor, 医歯学系, 教授 (70170800)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ITO Tatsuo Niigata University, Institute Medicine and Dentistry, Assistant, 医歯学系, 助手 (40345533)
|
|---|
| Project Period (FY) |
2004 – 2005
|
| Keywords | medaka / congenital malformation / ENU / positional cloning |
|---|
| Research Abstract |
To obtain the mutants with either brain or cardiovascular malformations, ENU mutagenesis of the medaka strain Hd-rR was conducted according to Ishikawa's method (Fish Biol J Medaka 10:27-29, 1999). Approximately 100 independent mutants were obtained. More than 90% of the mutants exhibited skeletal malformations. A small percentage of the mutants showed eye and fin anomalies. Two mutants with brain anomalies but no mutants with cardiovascular malformations were obtained. We analyzed the mutants with brain anomalies for gene expression of Shh, Wnt, Bmp, Zic, Msx and so on by RT-PCR and in situ hybridization. No significant alteration of the gene expression was observed in the genes examined. To isolate the causative mutations in the mutants with brain anomalies, positional cloning was started. We mated heterozygous mutant males with HNI strain females and obtained F1 fish. F1 males were backcrossed with heterozygous mutant Hd-rR females to produce F2 mutants. We isolated genomic DNAs from F2 mutants and started gene mapping using AFLP method (Nucleic Acid Res 23:4407-4414, 1995). During the early step of gene mapping experiments, we lost both of the mutant lines with brain anomalies because of unknown lethal infectious disease. We had to go back to the step of mutant generation by ENU and could not complete the positional cloning experiments.
|
