Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution.

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named "EV Fingerprinting" that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity.

ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.

Advanced Technologies and Automation in mES Cell Workflow.

Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. To streamline and increase the efficiency in our ES cell pipeline and decrease the timeline for mouse models produced via ES cells, automation is introduced in the pipeline. Below, we describe a novel and effective approach utilizing ddPCR, dPCR, automated DNA purification, MultiMACS, and adenovirus recombinase combined screening workflow that reduces the time between therapeutic target identification and experimental validation.

Molecular examination of the endogenous opioid system in rhesus macaque monkeys with self-injurious behavior.

Self-injurious behavior (SIB) can lead to serious injury and occurs in approximately 1%-4% of the adult population, with higher incidences in adolescent and institutionalized populations, as well as in children with developmental disorders such as Autism. SIB also spontaneously occurs in a low percentage of captive monkeys. Rhesus macaque (Macaca mulatta) monkeys are evolutionarily and physiologically similar to humans, share 93% genetic sequence similarity to humans, and have long been used as testing subjects for vaccine and clinical trials. Previous studies hypothesized that altered endogenous opioid expression occurs in the brains of individuals and animals that self-injure. We examined the regional mRNA expression of opioid signaling genes in sixteen rhesus macaques that exhibited SIB and eight sex- and age- matched controls. The brain regions examined are linked to reward reinforcement and stress adaptation including the hypothalamus, orbital frontal cortex, nucleus accumbens, hippocampus, caudate, and the amygdala. We found decreased µ-opioid receptor (OPRM1) in the amygdala of monkeys with SIB, and reduced prodynorphin (PDYN) in the hypothalamus. Our data suggest dysfunction in the regulation of opioid peptide precursors and calls for further investigation of the endogenous opioid system in SIB.