Optogenetics is an emerging technology for manipulating and controlling excitable tissues, such as the brain and heart. This technique requires genetic modification of cells to record light sensitivity. Photogenetic control of cardiomyocytes is achieved by genetic insertion of photosensitive ion channels and/or microbial derived (opsin) pumps to make cells controlled by light. The technology can be used for non-contact operation of activities in excitable tissues such as brain and heart.
Creative BioMart provides customized cardiomyocyte model construction services by infecting neonatal rat ventricular myocytes with channelrhodopsin-2 (ChR2) virus in vitro. We developed a procedure to quantitatively determine the optimal virus dose to obtain the best expression results. In addition, we can also provide the client with the service of optical driving of ChR2 transduced cells and measuring their functional response to light.
Microbial opsin, the core of optogenetics, can produce depolarization (excitation) current or hyperpolarization (inhibition) current in mammalian cells. Excitatory opsin, such as channel rhodopsin (ChR), can provide fast dynamic current of sufficient amplitude to trigger action potential.
When opsin is activated by photons of appropriate wavelength light, all trans retinoic aldehyde, a chromophore covalently bound to the channel, isomerizes to 13-cis-retinal, resulting in the opening of the channel. Depending on the channel, cations (ChR, AR) or anions (HR) flow with electrochemical gradients on the cell membrane or are pumped to change the transmembrane potential. Mutant stereopsin can also be designed in light sensitivity, speed and spectral response.
In the case of cardiac optogenetics, the most commonly used mutant excitatory opsin is ChR2(H134R). ChR2(H134R) is one of the earliest single amino acid mutants, resulting in a 2-3-fold increase in channel conductance with minimal effect on kinetics.
We infected neonatal rat ventricular cardiomyocytes (NRVM) in vitro with adenovirus containing ChR2 (H134R) gene. ChR2 (H134R) gene was fused with eYFP reporter protein with ubiquitous CMV promoter. The technology is also applicable to (and has been used in) other heart cell types, including adult cells, with properly designed photogenetic viruses and optimized virus infection protocols.
| Step | Service Description |
| Isolating and Preparing Cells for Adenoviral Infection | Cells are isolated and cultured through our proprietary technology |
| Determining the Optimal Multiplicity of Infection | Cardiomyocytes with opsin gene were infected with virus by suspension method, and the drug was administered appropriately while maintaining cell viability to achieve maximum efficiency. |
| Patterned Transgene Expression | The ability to infect NRVM cells in suspension allows patterned photogenetic transgene expression. Optogenetic actuation and feedback |
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