Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin - calcineurin - NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin - calmodulin Kinase - CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose-induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway.

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin–calcineurin–NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin–calmodulin kinase–CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.

Genomic safe harbors (GSHs) provide ideal integration sites for generating transgenic organisms and cells and can be of great benefit in advancing the basic and applied biology of a particular species. Here we report the identification of GSHs in a dry-preservable insect cell line, Pv11, which derives from the sleeping chironomid, Polypedilum vanderplanki, and similar to the larvae of its progenitor species exhibits extreme desiccation tolerance. To identify GSHs, we carried out genome analysis of transgenic cell lines established by random integration of exogenous genes and found four candidate loci. Targeted knock-in was performed into these sites and the phenotypes of the resulting transgenic cell lines were examined. Precise integration was achieved for three candidate GSHs, and in all three cases integration did not alter the anhydrobiotic ability or the proliferation rate of the cell lines. We therefore suggest these genomic loci represent GSHs in Pv11 cells. Indeed, we successfully constructed a knock-in system and introduced an expression unit into one of these GSHs. We therefore identified several GSHs in Pv11 cells and developed a new technique for producing transgenic Pv11 cells without affecting the phenotype.

Pv11 is the only animal cell line that can be preserved in the dry state at room temperature. Pv11 was derived from embryos of the sleeping chironomid Polypedilum vanderplanki, which displays an extreme form of desiccation tolerance known as anhydrobiosis. Pre-treatment with a high concentration of trehalose for 48 h allows Pv11 cells to enter anhydrobiosis. In the dry state, Pv11 cells preserve transgenic luciferase while retaining its activity; thus, these cells could be utilized as a vessel for dry-preserving valuable biological materials without loss of activity. However, Pv11 cells grow in suspension, which limits their applicability; for instance, they cannot be integrated into microfluidic devices or used in devices such as sensor chips. Therefore, in this paper, we sought to develop an effective immobilization system for Pv11 cells that, crucially, allows them to maintain their anhydrobiotic potential even when immobilized. First, we examined the effectiveness of various immobilization systems commonly used in standard cell cultures and found that Pv11 cells exhibited a very high adhesion rates with both biocompatible anchor for membrane (BAM) and Cell-Tak coatings. We also found that Pv11 cells immobilized well to uncoated glass if handled in serum-free medium. Next, we investigated whether immobilized Pv11 cells could retain their anhydrobiotic ability. While trehalose treatment of Pv11 cells prior to immobilization allowed them to retain a high level of both desiccation tolerance and proliferative potential after rehydration, trehalose treatment of Pv11 cells after immobilization resulted in a significant decrease in desiccation tolerance. Thus, it is important to induce anhydrobiosis before immobilization. In summary, we report the successful development of a protocol for the dry preservation of immobilized Pv11 cells.