Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics.
BACKGROUND
KillerRed (KR) is a novel photosensitizer that efficiently generates reactive oxygen species (ROS) in KR-expressing cells upon intense green or white light illumination in vitro, resulting in damage to their plasma membrane and cell death.


RESULTS
We report an in vivo modification of this technique using a fluorescent microscope and membrane-tagged KR (mem-KR)-expressing transgenic zebrafish. We generated several stable zebrafish Tol2 transposon-mediated enhancer-trap (ET) transgenic lines expressing mem-KR (SqKR series), and mapped the transposon insertion sites. As mem-KR accumulates on the cell membrane and/or Golgi, it highlights cell bodies and extensions, and reveals details of cellular morphology. The photodynamic property of KR made it possible to damage cells expressing this protein in a dose-dependent manner. As a proof-of-principle, two zebrafish transgenic lines were used to affect cell viability and function: SqKR2 expresses mem-KR in the hindbrain rhombomeres 3 and 5, and elsewhere; SqKR15 expresses mem-KR in the heart and elsewhere. Photobleaching of KR by intense light in the heart of SqKR15 embryos at lower levels caused a reduction in pumping efficiency of the heart and pericardial edema and at higher levels - in cell death in the hindbrain of SqKR2 and in the heart of SqKR15 embryos.


CONCLUSIONS
An intense illumination of tissues expressing mem-KR affects cell viability and function in living zebrafish embryos. Hence, the zebrafish transgenics expressing mem-KR in a tissue-specific manner are useful tools for studying the biological effects of ROS.
Institute of Molecular and Cell Biology, A-STAR, Singapore.
Cathleen
C
Teh
Dmitry M
DM
Chudakov
Kar-Lai
KL
Poon
Ilgar Z
IZ
Mamedov
Jun-Yan
JY
Sek
Konstantin
K
Shidlovsky
Sergey
S
Lukyanov
Vladimir
V
Korzh
D000818:Animals; D030801:Animals, Genetically Modified / Q000033:anatomy & histology / Q000196:embryology / Q000235:genetics; D017209:Apoptosis; D004251:DNA Transposable Elements; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology / Q000187:drug effects / Q000502:physiology; D004742:Enhancer Elements, Genetic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008856:Microscopy, Fluorescence / Q000379:methods*; D009024:Morphogenesis / Q000187:drug effects; D017319:Photosensitizing Agents / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000494:pharmacology; D015027:Zebrafish / Q000033:anatomy & histology / Q000196:embryology / Q000235:genetics
D004251:DNA Transposable Elements; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species; C508771:killer red protein, Anthomedusae; D049452:Green Fluorescent Proteins