Sherwood, J. | Search bioRxiv

bioRxiv2019

Localized delivery and uncaging of glutamate from MRI-visible albumin nanoclusters in the rat hippocampus using focused ultrasound

There is an ongoing need for noninvasive tools to manipulate brain activity with molecular, spatial and temporal specificity. Here we have investigated the use of MRI-visible, albumin-based nanoclusters for noninvasive, localized and temporally specific drug delivery to the rat brain. We demonstrated that IV injected nanoclusters could be deposited into target brain regions via focused ultrasound facilitated blood brain barrier opening. We showed that nanocluster location could be confirmed in vivo with MRI. Additionally, following confirmation of nanocluster delivery, release of the nanocluster payload into brain tissue can be triggered by a second focused ultrasound treatment performed without circulating microbubbles. Release of glutamate from nanoclusters in vivo caused enhanced c-Fos expression, indicating that the loading capacity of the nanoclusters is sufficient to induce neuronal activation. This novel technique for noninvasive stereotactic drug delivery to the brain with temporal specificity could provide a new way to study brain circuits in vivo preclinically with high relevance for clinical translation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=36 SRC="FIGDIR/small/696237v4_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b528f9org.highwire.dtl.DTLVardef@1950228org.highwire.dtl.DTLVardef@1c0ada0org.highwire.dtl.DTLVardef@c71790_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Pervasive head-to-tail insertions of DNA templates mask desired CRISPR/Cas9-mediated genome editing events

CRISPR/Cas9 mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or non-homologous end-joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis--in most cases--failed to identify such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential, and offer practical solutions to correctly identify precisely edited chromosomes.

genetics↗