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Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

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Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring.Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Dialysis patients and kidney transplant (KTX) recipients suffer from an impaired immune system and show a decreased response to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination. We performed a retrospective analysis of 1505 serological SARS-CoV-2 measurements obtained from 887 dialysis patients and 86 KTX recipients. The results were separated by patient subgroups (dialysis/KTX) as well as SARS-CoV-2 status. The latter criterion included SARS-CoV-2-naïve patients with or without COVID-19 vaccination and convalescent patients receiving a booster shot. Serologies of 27 vaccinated healthy individuals served as the reference group. Vaccine-induced cellular immune response was quantified by an interferon-γ release assay in 32 KTX recipients. We determined seroconversion rates of 92.6%, 93.4%, and 71.4% in dialysis patients vaccinated with either BNT162b2, mRNA-1273, or AZD1222, respectively. Vaccination-induced anti-SARS-CoV-2 antibody titers were lower in dialysis patients compared to healthy individuals, and vaccination with mRNA-1273 induced higher titers than BNT162b2. The initial seroconversion rate was 39.5% in KTX recipients vaccinated with BNT162b2. A linear regression model identified medication with mycophenolate-mofetil/mycophenolic acid as an independent risk factor for missing seroconversion. Within a cohort of 32 KTX recipients, cellular and humoral immune reactivity to SARS-CoV-2 was detectable in three patients only. Conclusively, vaccine-induced seroconversion rates were similar in dialysis patients compared to healthy individuals but were strongly impaired in KTX recipients. Anti-SARS-CoV-2 IgG titers elicited by double active immunization were significantly lower in both cohorts compared to healthy individuals, and immune responses to vaccination vanished quickly.

Background: Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. Methods: We here apply previously published sample preparation methods together with STED and confocal microscopy for resolving nanoscale podocyte substructure. The protocols are modified and optimized in order to be applied to samples which have been formalin fixed and paraffin-embedded (FFPE). Results: We successfully modify our protocols to allow for deep three-dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. We further show that quantitative analysis can be applied to extract morphometrics of healthy and diseased samples from both mice and humans. Conclusions: The results from this study could increase the feasibility to implement optical kidney imaging protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the glomerular capillary surface area available for filtration and alter the intrinsic permeability of the capillary wall resulting in albuminuria, however, direct quantitative assessment of the underlying morphological changes has not been possible so far. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with super-resolved stimulated emission depletion (STED) microscopy or confocal microscopy. Our method, Automatic Morphological Analysis of Podocytes (AMAP), detected 87-95% manually-annotated foot processes and additionally recognized 1.3 - 2.17-fold more. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm was applied to a set of human kidney disease conditions allowing comprehensive quantifications of various underlying morphometric parameters. These data confirmed that when podocytes are injured, they take on a more simplified architecture and the slit-diaphragm length is much reduced, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the GBM to albumin.