Journal of Neurogastroenterology and Motility : eISSN 2093-0887 / pISSN 2093-0879

Download original image
Fig. 3. Schematic overview of possible strategies to deliver optical probes (synthetic and genetic) into intestinal tissues. The top row shows 3 methods to apply small synthetic dyes to ganglia, interstitial cells, and muscle layers. During bulk loading tissues are incubated in a buffer containing an AM-ester of a Ca2+ indicator (common examples are: Indo-1, Fluo-3, and Fluo-4?), Oregon Green BAPTA, Rhod-2, etc). The esters are cleaved by intracellular esterases, whereby the indicator becomes functional and is trapped within the cell. With bulk loading, the outermost layers will have higher levels of dye than the inside layers. Using sharp (or patch) electrodes Ca2+ indicators can also be loaded in individual cells,, or alternatively, dyes can be applied locally as often done with di-8-ANEPPS to reduce labeling of other layers in the field of view. Strategies to express genetically encoded proteins mostly depend on the technology to deliver the coding DNA into the cells of interest. Since simple transfection methodology cannot be used in tissues, knockin or transgenic animals often with binary expression systems based on recombination (Cre-loxP) or transactivation technology need to be used. Here, the main determinant of protein expression is the specificity and strength of the promoter/enhancers. In case of binary expression systems, a ubiquitous promoter (eg, cytomegalovirus) can be used to optimize expression levels while cellular specificity is achieved by the control element driving Cre recombinase. Apart from transgenic animals, viral approaches can also be used either by injecting viral vector in the bloodstream or by delivering vector intraluminally. Here the combination of viral tropism and cell type specific promoters can help to yield expression in a subset of intestinal cells. For a comprehensive overview of genetic approaches that can be used to target specific cell types we refer to an excellent review by Huang and Zeng.
J Neurogastroenterol Motil 2015;21:337~351
© J Neurogastroenterol Motil