Optogenetic in vivocell manipulation in KillerRed-expressing zebrafish transgenics

Cathleen Teh,Sergey Lukyanov,Ilgar Z Mamedov,Jun-Yan Sek,Konstantin Shidlovsky,Vladimir Korzh,Dmitry M Chudakov,Kar-Lai Poon

doi:10.1186/1471-213x-10-110

Abstract

BackgroundKillerRed (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.ResultsWe 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.ConclusionsAn 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.

Highlights

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
The Tol2-KR screen To test whether the KR would remain an efficient photosensitizer in developing zebrafish embryos, the cytosolic green fluorescent protein (GFP) reporter in the Tol2 transposon pBKCMV enhancer trap (ET) vector [17], carrying a partial epithelial promoter of the keratin4 gene, was replaced by the membrane-tethered version of KR
Injected embryos were raised to sexual maturity when, as adults, they were outcrossed with wild type fish to identify transgenic progeny (F1) with tissue-specific expression of KR

Summary

Introduction

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. The introduction of efficient transgenesis into the field of developmental biology opened the possibility to eradicate cells through the incorporation of tissue-specific and inducible toxic proteins [1,2,3,4], with cell death as an experimental endpoint. The level of ROS generated can be modulated by a dose of illumination and evaluated by photobleaching of fluorescent proteins [5]. Using purified KR and chemical probes to detect superoxide and singlet oxygen, it was shown that both types of ROS were produced upon green light irradiation of KR-expressing cells in vitro [12,13,14,15]

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