Gene Editing in Rat Embryonic Stem Cells to Produce In Vitro Models and In Vivo Reporters

Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats.This research was funded by the European Community project EURATRANS (grant no. HEALTH-F4-2010-241504), the Biotechnology and Biological Sciences Research Council of the United Kingdom (grant no. BB/H012737/1), the Swiss National Science Foundation Sinergia Program, the Louis-Jeantet Foundation, and the Isaac Newton Trust. R.T.K. was supported by a Wellcome Trust Research Career Development Fellowship (grant no. 091543/Z/10/Z) and a Lister Institute Research Prize. A.S. is a Medical Research Council Professor (grant no. G1100526/1)

Second Cheese Whey Treatment Using Zeolite under Continuous Flow Mode and Its Application on Wheat Growth

The efficiency of natural zeolite to treat second cheese whey (SCW) and remove ammonium from artificial wastewater (AWW) was examined. Since zeolite has been reported to improve nitrogen availability in soils, its effect on wheat plant growth was also examined. Continuing a previous study using batch reactors, results are presented concerning experiments in fixed-bed columns under continuous operation. Results from the continuous flow column experiments using AWW and zeolite (2.0−2.8 mm) indicated that low flow rates (4 mL/min and 8 mL/min) did not significantly affect zeolite adsorption ability, while maximum zeolite adsorption capacity reached 15.30 mg NH4+-N/g. Finally, the effect of zeolite saturated with NH4+-N on plant growth was examined. The application of saturated zeolite affected significantly wheat plant growth and resulted in faster growth and higher biomass production

Surpassing light-induced cell damage in vitro with novel cell culture media

Light is extensively used to study cells in real time (live cell imaging), separate cells using fluorescence activated cell sorting (FACS) and control cellular functions with light sensitive proteins (Optogenetics). However, photo-sensitive molecules inside cells and in standard cell culture media generate toxic by-products that interfere with cellular functions and cell viability when exposed to light. Here we show that primary cells from the rat central nervous system respond differently to photo-toxicity, in that astrocytes and microglia undergo morphological changes, while in developing neurons and oligodendrocyte progenitor cells (OPCs) it induces cellular death. To prevent photo-toxicity and to allow for long-term photo-stimulation without causing cellular damage, we formulated new photo-inert media called MEMO and NEUMO, and an antioxidant rich and serum free supplement called SOS. These new media reduced the detrimental effects caused by light and allowed cells to endure up to twenty times more light exposure without adverse effects, thus bypassing the optical constraints previously limiting experiments.Funding was provided by grants from the Wellcome Trust (091543/Z/10/Z), Isaac Newton Trust and The Royal Society

Zeolite as a Potential Medium for Ammonium Recovery and Second Cheese Whey Treatment

The efficiency of natural zeolite to remove ammonium from artificial wastewater (ammonium aqueous solutions) and to treat second cheese whey was examined, aiming to recover nitrogen nutrients that can be used for further applications, such as slow-release fertilizers. Sorption experiments were performed using artificial wastewater and zeolite of different granulometries (i.e., 0.71–1.0, 1.8–2.0, 2.0–2.8, 2.8–4.0, and 4.0–5.0 mm). The granulometry of the zeolite had no significant effect on its ability to absorb ammonium. Nevertheless, smaller particles (0.71–1.0 mm) exhibited quicker NH4+-N adsorption rates of up to 93.0% in the first 10 min. Maximum ammonium removal efficiency by the zeolite was achieved at ammonium concentrations ranging from 10 to 80 mg/L. Kinetic experiments revealed that chemisorption is the mechanism behind the adsorption process of ammonium on zeolite, while the Freundlich isotherm model fitted the experimental data well. Column sorption experiments under batch operating mode were performed using artificial wastewater and second cheese whey. Column experiments with artificial wastewater showed high NH4+-N removal rates (over 96% in the first 120 min) for all granulometries and initial NH4+-N concentrations tested (200 and 5000 mg/L). Column experiments with second cheese whey revealed that natural zeolite can remove significant organic loads (up to 40%, 14.53 mg COD/g of zeolite) and NH4+-N (about 99%). For PO43−-P, the zeolite appeared to saturate after day 1 of the experiments at a removal capacity of 0.15 mg P/g of zeolite. Desorption experiments with water resulted in low NH4+-N and PO43−-P desorption rates indicating that the zeolite could be used as a substrate for slow nitrogen release in soils