Xenopus laevis Model for Optogenetic Research

KillerRed (KR) is a recently discovered fluorescent protein. When activated by green light, it will release reactive oxygen species (ROS) into the cytoplasm, resulting in apoptosis of KR expressing cells. This characteristic allows the use of Kr as a means of killing cells with great temporal and spatial specificity in organisms, while minimizing the nonspecific effects that may be caused by mechanical or chemical exposure injury techniques.

Creative BioMart developed an expression protocol that targets ablated tissue by light induced cell death in toad tadpoles expressing KR. We microinjected membrane bound Kr mRNA into Xenopus embryos to express KR in regions of interest for our customers. The customer can expose the animal body to green light under the fluorescence microscope to induce apoptosis in the target tissue.

Light Induced Apoptosis to Xenopus laevis Model

How organisms repair damaged tissues and regenerate missing tissues, sometimes even whole organs and appendages, has always been a prominent problem in biology. Xenopus laevis has also been proved to be an effective and popular experimental model system for regeneration and repair. In order to study the regeneration or repair of specific organs or tissues, it is necessary to destroy or remove target cells, sometimes with high specificity, in order to limit the impact of injury on target organs and subsequent repair reactions or tissues. Compared with traditional techniques, light induced cell death has many advantages, including greater temporal and spatial specificity and corresponding reduction of off target effect.

Expression Protocol at Creative BioMart

KR is a dimeric red fluorescent protein with excitation / emission maximum at 585 / 610 nm. When excited by green light, it will release reactive oxygen species (ROS) in the form of superoxide and singlet oxygen. After exposure to green light, Kr expressing cells die through apoptosis, which either directly activates the apoptotic pathway through mitochondrial Kr or binds phospholipids in Kr oxidized plasma membrane. In addition, Kr targeting the nuclear layer or histone can induce DNA breakage and stop the cell cycle

We will construct the plasmid according to the selected KR expression site. We induced ovulation for adult female Xenopus laevis by injection of chorionic gonadotropin and fertilized through standard pathway.
Embryos were injected at the cleavage stage using a microinjector and a capillary glass needle. After injection, we will test the subjects for KR activation and ROS.
After confirming the correct expression of KR at the target location, we also provided immunohistochemistry and in situ hybridization and expression measurement.

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