1. Optogenetic Therapeutic Cell Implants
David Ausländer, Martin Fussenegger 
Gastroenterology 
Volume 143, Issue 2, Pages (August 2012)
DOI: /j.gastro
Copyright © 2012 AGA Institute Terms and Conditions

2. Figure 1
Synthetic phototransduction pathway resulting in GLP-1 expression. A synthetic phototransduction pathway in mammalian cells is embedded into the natural cellular system. (1) The blue light-responsive module comprises a constitutive expression unit producing melanopsin, which is located at the plasma membrane and binds 11-cis-retinal as a chromophore. Upon illumination, conformational changes within the protein lead to the activation of (2) an endogenous, Gaq-dependent signaling cascade. The activation of phospholipase C (PLC) and protein kinase C (PKC) leads to intracellular calcium release from transient receptor potential channels (TRPC) and possibly also from cellular storage locations such as the endoplasmic reticulum (ER). As a calcium-binding protein, calmodulin associates with calcineurin that dephosphorylates the transcription factor nuclear factor of activated T cells (NFAT), which subsequently translocates into the nucleus. (3) NFAT activates its cognate promoter, which drives GLP-1 expression that is encoded by the reporter module. (4) Finally, GLP-1 is secreted into the extracellular matrix with its capacity to act as a potential therapeutic protein in vivo.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

3. Figure 2
Engineering a light-responsive therapeutic cell implant: A schematic, 5-step illustration. Step 1: To achieve customized expression levels of functional parts within mammalian cells, synthetic biologists can access a huge cellular toolbox. Promoter and operator sequences, combined with tailor-made transcription factors, have enabled controlled gene expression in mammals.33 Furthermore, RNA molecules like aptamers or ribozymes have been successfully shown to influence translation34 or transcription35 of mRNAs encoding proteins with various functions. Step 2: In the next step, assembled constructs are transfected into mammalian cells, such as human embryonic kidney cells (HEK-293) or Chinese hamster ovary cells (CHO-K1), after their functionality has been validated in cell culture. Usually, a rigorous characterization of the artificial network performing a task of interest follows to allow improved predictability of the system's behavior. Step 3: In therapeutic applications, engineered cells are implanted into the host organism. Polymer capsules (consisting of alginate-poly-(L)-lysine-alginate or cellulose sulfate) or hollow fiber membrane containers have been used to shield the cells with a semipermeable and biocompatible layer that enables diffusion of nutrients and biomolecules while simultaneously protecting the cells from the host immune system. These cell-containing devices have been successfully placed subcutaneously or intraperitoneally into mice. Step 4: Therapeutic cell implants can either be induced exogenously or sense an endogenous metabolite, thereby acting as a sensor-effector device. Different exogenous stimuli, ranging from chemicals to biomolecules, have been efficiently applied, but light stands out as a noninvasive, adjustable, and easy-to-use input signal. Importantly, the presence of the therapeutic protein in the serum of mice has to be confirmed by quantitative biochemical methods. Step 5: Finally, the therapeutic applicability of the optogenetic cell implant is verified by using appropriate animal models.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions