Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

BACKGROUND: The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. RESULTS: We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. CONCLUSIONS: Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain

microPREP: A new laser tool for high-volume sample preparation

Over the past fifty year, lasers have perpetuated to find new, often groundbreaking applications in science and technology. The most important features of lasers are that photons are inherently free of elemental contamination, extremely high energy densities can be focused in very small areas and the laser beam can be precisely positioned using deflection mirrors. By reducing pulse lengths from a few nanoseconds down to the picosecond or femtosecond range, material’s ablation is becoming increasingly "athermal", i.e. structure damage by local heating is reduced to well below a few microns. In view of these outstanding characteristics of lasers as tools for micromachining, it is very surprising that sample preparation for microstructure diagnostics so far hasn’t made use of laser technology. microPREPTM, the all-new, patented laser-micromachining tool developed by 3D-Micromac is the first instrument to make fast, clean, and efficient laser ablation available for the preparation of samples for microstructure diagnostics. Exemplified for a sample to be investigated by transmission electron microscopy (TEM) and following a three-stage approach, a supporting basic structure is cut from the feedstock first. Second, the supported structure is thinned down to a few micron of residual thickness and third, the supported and thinned structure is polished using an ion broad beam. Illustrated by numerous examples, it is shown that this technology is ready to be applied on different areas of microstructure diagnostics and has very high potential for failure diagnostics

Routine monitoring of sodium and phosphorus removal in peritoneal dialysis (PD) patients treated with continuous ambulatory PD (CAPD), automated PD (APD) or combined CAPD plus APD.

Background: Adequate removal of sodium (Na) and phosphorus (P) is of paramount importance for patients with dialysis-dependent kidney disease can easily quantified in peritoneal dialysis (PD) patients. Some studies suggest that automated PD (APD) results in lower Na and P removal. Methods. In this study we retrospectively analysed our data on Na and P removal in PD patients after implementation of a routine monitoring in 2011. Patients were stratified in those treated with continuous ambulatory PD (CAPD, n=24), automated PD (APD, n=23) and APD with one bag change (CAPD+APD, n=10). Until 2015 we collected time-varying data on Na and P removal from each patient (median 5 [interquartile range 4-8] values). Results: Peritoneal Na and P removal (mmol per 24h +/- standard deviation) was 102 +/- 48 and 8 +/- 2 in the CAPD, 90 +/- 46 and 9 +/- 3 in the APD and 126 +/- 39 and 13 +/- 2 in the CAPD+APD group (ANOVA P=0.141 and <0.001). Taking renal excretion into account total Na and P removal (mmol per 24h) was 221 +/- 65 and 16 +/- 5 in the CARD, 189 +/- 58 and 17 +/- 6 in the APD and 183 +/- 38 and 16 +/- 6 in the CAPD+APD group (P=0.107 and 0.764). Over time, peritoneal removal of Na but not that of P increased in all groups. In patients with modifications of PD treatment, Na but not P removal was significantly increased over-time. Conclusions: Overall Na and P removal were similar with different PD modalities. Individualized adjustments of PD prescription including icodextrin use or higher glucose concentration can improve Na removal while P removal is mainly determined by the dialysate volume

Kidney-specific inactivation of the megalin gene impairs trafficking of renal inorganic sodium phosphate cotransporter (NaPi-IIa).

Renal reabsorption of inorganic phosphate is mediated by the type IIa sodium phosphate cotransporter (NaPi-IIa) of the proximal tubule. Changes in renal phosphate handling are mainly attributable to altered NaPi-IIa brush border membrane (BBM) expression. Parathyroid hormone (PTH) induces inactivation of NaPi-IIa by endocytic membrane retrieval and degradation. The key elements triggering this process are not clear to date. Megalin serves as a receptor for the endocytosis of multiple ligands and is coexpressed with NaPi-IIa in the proximal tubule. Investigated was the role of megalin in the regulation of NaPi-IIa in steady state and during inactivation. Kidneys and tubular BBM fractions from mice with a renal-specific megalin gene defect and from controls were analyzed by light and electron microscopic histochemical techniques and Western blot test. Steady-state levels of NaPi-IIa in BBM were significantly enhanced, mRNA levels preserved, and phosphaturia reduced in the absence of megalin. Fluid-phase endocytosis was prevented and the apical endocytic apparatus markedly reduced. Systemic administration of PTH resulted in a defective retrieval and impaired degradation of NaPi-IIa. In vitro, the application of various stimuli of the PTH-induced signaling cascade had no effect either. Adequate steady-state expression of NaPi-IIa and the capacity of the proximal tubule cell to react on PTH-driven inactivation of NaPi-IIa by endocytosis and intracellular translocation require the presence of megalin