Establishment of the expression system for studying the function of active caspase-3 in zebrafish.
Caspase-3, a key molecule in apoptosis, has been extensively studied in cell culture system; however, it has been less well characterized in vivo because certain mediators are required for the proteolytic activation of effector caspases, including caspase-3. In this study, various forms of caspase-3 with the C-terminal GFP tag were inserted into the pCS2+ plasmid, and the expression patterns of caspase-3 proteins were characterized in a zebrafish model system using microinjection of nucleic acids into zebrafish embryos. We have verified that active caspase-3 was generated by its autocatalytic activity under the condition of caspase-2 prodomain (C2P)-caspase-3-GFP overexpression, indicating that the C2P domain is crucial for the activation of caspase-3. We also confirmed that the C2P domain plays an important role in regulating the nuclear localization of the C2P-caspase-3 chimeric protein. We used this expression system to establish an animal model system suitable for the investigation of the functional characteristics of caspase-3 in vivo. Thus, our study provides a useful and specific tool for investigating the molecular mechanisms by which active caspase-3 regulates apoptosis during embryonic development.
Kim
Ho-Young
HY
Department of Biomedical Sciences, The Catholic University of Korea, Seoul 137-701, Korea. hrhim@catholic.ac.kr
Kim
Goo-Young
GY
Kim
Sang-Soo
SS
Nam
Min-Kyung
MK
Rhim
Hyangshuk
H
D021581:Active Transport, Cell Nucleus; D000818:Animals; D053148:Caspase 3 / Q000235:genetics*; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000008:administration & dosage; D008845:Microinjections; D023421:Models, Animal; D010957:Plasmids / Q000008:administration & dosage; D014162:Transfection / Q000379:methods*; D015027:Zebrafish
D049452:Green Fluorescent Proteins; D053148:Caspase 3