An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

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

Live birth rate of offspring upon transgenesis by PNI or EEZy.

<p>Live birth rate of offspring upon transgenesis by PNI or EEZy.</p

Embryo toxicity of EEZy as compared to pronuclear injection.

<p>(A) Assessment of embryo development as percentage of developed blastocysts from zygotes after EEZy. Untreated zygotes are compared to zygotes electroporated with Opti-MEM (Mock) or CRISPR/Cas9 components targeting the <i>Gt(ROSA)26Sor</i> locus (CRISPR). Data represent three independent experiments. (B) Quantification of viable <i>Nphs2</i>-targeted zygotes from four experiments immediately after PNI compared to electroporated zygotes and (C) the correspondingly developed blastocysts. (D) Developed blastocyst calculated from the number of zygotes before transgenesis in (B). (E) Quantification of RFLP genotyping from four independent experiments for HDR efficiency in blastocysts from zygotes after PNI compared to electroporated zygotes from the experiments depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196891#pone.0196891.g001" target="_blank">Fig 1B and 1D</a>, right columns. Data are means ± standard deviation. *p < 0.05, **p < 0.01, ns = non-significant. N = total number of embryos analyzed.</p

Identification of Acer2 as a First Susceptibility Gene for Lithium-Induced Nephrogenic Diabetes Insipidus in Mice

BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identifie

Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell

Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity

Regulation of microRNA function in somatic stem cell proliferation and differentiation

microRNAs (miRNAs) are important modulators of development. Owing to their ability to simultaneously silence hundreds of target genes, they have key roles in large-scale transcriptomic changes that occur during cell fate transitions. In somatic stem and progenitor cells — such as those involved in myogenesis, haematopoiesis, skin and neural development — miRNA function is carefully regulated to promote and stabilize cell fate choice. miRNAs are integrated within networks that form both positive and negative feedback loops. Their function is regulated at multiple levels, including transcription, biogenesis, stability, availability and/or number of target sites, as well as their cooperation with other miRNAs and RNA-binding proteins. Together, these regulatory mechanisms result in a refined molecular response that enables proper cellular differentiation and function