Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles

Abstract

Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on its surface. Because of their central gold particle, SmartFlare probes are detectable by transmission electron microscopy. The aim of the present study was to investigate the uptake and distribution of SmartFlare probes in equine conceptuses at developmental stages suitable for embryo transfer (Days 6-10), equine trophoblast vesicles and equine dermal fibroblast cell cultures, and to determine whether differences among these cell types and structures exist. Probe uptake was followed by transmission electron microscopy and fluorescence microscopy. Although the embryonic zona pellucida did not reduce uptake of the probe, the acellular capsule fully inhibited probe internalisation. Nanogold particles were taken up by endocytosis by all cell types examined in a similar manner with regard to time and intracellular migration. They were processed in endosomal compartments and accumulated within lysosomal structures after longer incubation times. In conclusion, the SmartFlare probe is applicable in equine conceptuses, but its use is limited to the developmental stages before the formation of the embryonic capsule.