A Photoprotein in Mouse Embryonic Stem Cells Measures Ca2+ Mobilization in Cells and in Animals

Abstract

Exogenous expression of pharmacological targets in transformed cell lines has been the traditional platform for high throughput screening of small molecules. However, exogenous expression in these cells is limited by aberrant dosage, or its toxicity, the potential lack of interaction partners, and alterations to physiology due to transformation itself. Instead, primary cells or cells differentiated from precursors are more physiological, but less amenable to exogenous expression of reporter systems. To overcome this challenge, we stably expressed c-Photina, a Ca2+-sensitive photoprotein, driven by a ubiquitous promoter in a mouse embryonic stem (mES) cell line. The same embryonic stem cell line was also used to generate a transgenic mouse that expresses c-Photina in most tissues. We show here that these cells and mice provide an efficient source of primary cells, cells differentiated from mES cells, including cardiomyocytes, neurons, astrocytes, macrophages, endothelial cells, pancreatic islet cells, stably and robustly expressing c-Photina, and may be exploited for miniaturized high throughput screening. Moreover, we provide evidence that the transgenic mice may be suitable for ex-vivo bioimaging studies in both cells and tissues.

Histamine up-regulates fibroblast growth factor receptor 1 and increases FOXP2 neurons in cultured neural precursors by histamine type 1 receptor activation: conceivable role of histamine in neurogenesis during cortical development in vivo

“…Recently, mouse embryonic stem cells were engineered to express a Ca 2+ -sensitive protein, and HA was used to select clones that augmented [Ca 2+ ] i , confirming the presence of histaminergic G-protein coupled receptors in pluripotent stem cells. Furthermore, neural differentiation of these embryonic stem cells rendered cells that also were able to increase [Ca 2+ ] i after addition of 100 μM HA; all these effects were mediated by H 1 R [31], which is consistent with our findings.…”

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

“…We recently described the generation of PhotoTopo mice (Cainarca et al 2010). Briefly, c‐Photina – a photoprotein derived from Clytin ( Clytia gregaria ) and modified by random mutagenesis – was optimized for mammalian codon usage and fused to a mitochondrial tag (human cytochrome C oxidase, subunit VIII).…”

“…Here, we establish a live‐cell bioluminescence [Ca 2+ ] m imaging approach that overcomes these limitations without compromising its numerous advantages. Using genetically engineered PhotoTopo mice that ubiquitously express the optically improved photoprotein c‐Photina within the mitochondrial matrix (Cainarca et al 2010) and a dedicated bioluminescence microscope, we record (sub)cellular mitochondrial Ca 2+ signals at millisecond resolution. Combining both [Ca 2+ ] m and [Ca 2+ ] c imaging methods with whole‐cell patch‐clamp recordings, we describe here the key components of a Ca 2+ mobilization network that underlies purinergic signalling in primary mouse Sertoli cells.…”

HTS/HCS to Screen Molecules Able to Maintain Embryonic Stem Cell Self-Renewal or to Induce Differentiation: Overview of Protocols

“…A mES cell line expressing a Ca2 + −activated photoprotein as a reporter gene under the control of a ubiquitous promoter was generated [63]. Differentiating the cPhotina mES cell line into neuronal cells, it was demonstrated that this system is sensitive enough to study the pharmacological modulation of target genes that induce Ca2+ mobilization.…”