Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies-particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney.
Figure 1. KillerRed (KR)-mediated light-induced apoptosis results in ablated eye phenotype. Xenopus laevis embryos were injected with KR mRNA at NieuwkoopâFaber (NF) stage 2â3 and selected for KR expression in the left eye at NF stage 26â28.Notes: (A) Five minute activation of KR by exposure to green light induced photobleaching. (B, C) 1.5â3.5 hours after light treatment in eye (or at matching stages for unlit tadpoles), immunohistochemistry for active Caspase-3 showed that light activation of KR resulted in significantly higher levels of apoptosis over tadpoles not exposed to light, or expressing KR. (Fisherâs exact; *p < 0.01; **p < 0.001). Apoptosis was spatially restricted to the illuminated region. (D) After 24 hours, tadpoles were visually inspected for the presence of an ablated phenotype in the left eye. (E) Light activation of KR resulted in significantly higher incidence of ablated eye phenotype. (Fisherâs exact; *p < 0.001). All scale bars are 500 μm. n indicates the number of tadpoles across all replicates, and N indicates the number of replicates shown. (A, B: TRITC; D: Brightfield).
Figure 2. Photoactivation of KillerRed (KR) increases reactive oxygen species only within the illuminated region. Dihydroethidium (DHE) is a fluorescent indicator of superoxides. Following five minute activation of KR by exposure to green light, DHE fluorescence greatly increased within the illuminated region (dashed circle). However, it did not similarly increase in nearby, non-illuminated KR-expressing regions, like the forebrain and cement gland (arrowheads). All scale bars are 200 μm. (Top: TRITC; middle, bottom: Mermaid.)
Figure 3. KillerRed (KR)-mediated light-induced apoptosis results in ablated pronephros. Xenopus laevis embryos were injected with KR mRNA at Nieuwkoop–Faber (NF) stage 2–3 and selected for KR expression in the left pronephros at NF stage 37.Notes: (A) Five minute activation of KR by exposure to green light induced photobleaching in pronephros (circled). (B, C) 1.5–3.5 hours after light treatment in pronephros (or at matching stages for unlit tadpoles), immunohistochemistry for active Caspase-3 showed that light activation of KR resulted in a significant increase in the number of Caspase-positive loci over tadpoles not exposed to light, or expressing KR (Tukey–Kramer; *p < 0.001). Mean number of Caspase-positive loci per tadpolepronephros is shown, with error bars indicating the standard error of the mean. Apoptosis was spatially restricted to the illuminated region. (D) After 24 hours, in situ hybridization for senescence marker protein 30 (SMP30) was performed to visualize proximal tubules, and areas of SMP30 expression were measured on each side of the tadpole using SPOT Advanced imaging software. (E) Light activation of KR resulted in ablation of proximal tubules on the left side, compared to tadpoles with KR expression alone and tadpoles with no treatment at all. For (B) (inset) scale bars are 100 μm; all other scale bars are 500 μm. n indicates the number of tadpoles across all replicates, and N indicates the number of replicates shown. (A, B: TRITC; D: Brightfield.)
Beck,
Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms.
2009, Pubmed,
Xenbase
Beck,
Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms.
2009,
Pubmed
,
Xenbase
Birnbaum,
Slicing across kingdoms: regeneration in plants and animals.
2008,
Pubmed
Bulina,
A genetically encoded photosensitizer.
2006,
Pubmed
Bulina,
Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed.
2006,
Pubmed
Caine,
Regeneration of functional pronephric proximal tubules after partial nephrectomy in Xenopus laevis.
2013,
Pubmed
,
Xenbase
Deisseroth,
Optogenetics.
2011,
Pubmed
DENT,
Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad.
1962,
Pubmed
FREEMAN,
LENS REGENERATION FROM THE CORNEA IN XENOPUS LAEVIS.
1963,
Pubmed
,
Xenbase
GLUCKSMANN,
Cell deaths in normal vertebrate ontogeny.
1951,
Pubmed
Hayakawa,
Inhibition of cardiac myocyte apoptosis improves cardiac function and abolishes mortality in the peripartum cardiomyopathy of Galpha(q) transgenic mice.
2003,
Pubmed
Hensey,
Programmed cell death during Xenopus development: a spatio-temporal analysis.
1998,
Pubmed
,
Xenbase
Jacobson,
Programmed cell death in animal development.
1997,
Pubmed
Matsushita,
Interleukin-6/soluble interleukin-6 receptor complex reduces infarct size via inhibiting myocardial apoptosis.
2005,
Pubmed
Moody,
Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.
1990,
Pubmed
,
Xenbase
Nishikawa,
Spatial, temporal and hormonal regulation of programmed muscle cell death during metamorphosis of the frog Xenopus laevis.
1995,
Pubmed
,
Xenbase
Palojoki,
Cardiomyocyte apoptosis and ventricular remodeling after myocardial infarction in rats.
2001,
Pubmed
Qi,
Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG.
2012,
Pubmed
Serebrovskaya,
Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein.
2009,
Pubmed
Serebrovskaya,
Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein.
2011,
Pubmed
,
Xenbase
Shibuya,
Deleterious effects of mitochondrial ROS generated by KillerRed photodynamic action in human cell lines and C. elegans.
2012,
Pubmed
Shirmanova,
Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice.
2013,
Pubmed
Shu,
A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.
2011,
Pubmed
Teh,
Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics.
2010,
Pubmed
Tseng,
Apoptosis is required during early stages of tail regeneration in Xenopus laevis.
2007,
Pubmed
,
Xenbase
Vegh,
Reactive oxygen species in photochemistry of the red fluorescent protein "Killer Red".
2011,
Pubmed
Vergara,
Retinal regeneration in the Xenopus laevis tadpole: a new model system.
2009,
Pubmed
,
Xenbase
Waldeck,
Positioning effects of KillerRed inside of cells correlate with DNA strand breaks after activation with visible light.
2011,
Pubmed
