Targeted single-cell gene induction by optimizing the dually regulated CRE/loxP system by a newly defined heat-shock promoter and the steroid hormone in Arabidopsis thaliana.

Multicellular organisms rely on intercellular communication systems to organize their cellular functions. In studies focusing on intercellular communication, the key experimental techniques include the generation of chimeric tissue using transgenic DNA recombination systems represented by the CRE/loxP system. If an experimental system enables the induction of chimeras at highly targeted cell(s), it will facilitate the reproducibility and precision of experiments. However, multiple technical limitations have made this challenging. The stochastic nature of DNA recombination events, especially, hampers reproducible generation of intended chimeric patterns. Infrared laser-evoked gene operator (IR-LEGO), a microscopic system that irradiates targeted cells using an IR laser, can induce heat shock-mediated expression of transgenes, for example, CRE recombinase gene, in the cells. In this study, we developed a method that induces CRE/loxP recombination in the target cell(s) of plant roots and leaves in a highly specific manner. We combined IR-LEGO, an improved heat-shock-specific promoter, and dexamethasone-dependent regulation of CRE. The optimal IR-laser power and irradiation duration were estimated via exhaustive irradiation trials and subsequent statistical modeling. Under optimized conditions, CRE/loxP recombination was efficiently induced without cellular damage. We also found that the induction efficiency varied among tissue types and cellular sizes. The developed method offers an experimental system to generate a precisely designed chimeric tissue, and thus, will be useful for analyzing intercellular communication at high resolution in roots and leaves.

Comparison of Areal Productivity of Nannochloropsis oceanica Between Lab-Scale and Industrial-Scale Raceway Pond.

The areal biomass productivities (g-1 m-2 day-1) of Nannochloropsis oceanica between different sizes of way ponds were compared. Sequential batch cultivation using 2-m2, 20-m2, and 200-m2 raceway ponds with an industrial scale 4000-m2 raceway as the main culture was conducted in summer and autumn during 2017 at Whyalla, Australia. Areal productivities of sequential batch cultivation during the same culture period were 8.4 g ± 0.9 g-1 m-1 day-1 in the 2-m2 ponds, 9.3 g-1 m-1 day-1 in the 20-m2 ponds, and 8.0 g-1 m-1 day-1 in the 200-m2 ponds respectively. In parallel with the operation of the main 4000-m2 pond, some smaller scale ponds of 2, 20, and 200 m2 were operated at the same site under the same conditions. Areal productivity data of dry biomass of Nannochloropsis oceanica in each pond are very similar between industrial 4000-m2 pond and other smaller ponds. In this work, the authors demonstrate that using the growth rate and productivities of Nannochloropsis from smaller scale open ponds with the same depth is valid to estimate for large-scale ponds in excess of 4000 m2.