Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

LAGLIDADG homing endonucleases (LHEs) cleave 18–24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE–dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities

Barcoding cells using cell-surface programmable DNA-binding domains

We report an approach to barcode cells through cell-surface expression of programmable zinc-finger DNA-binding domains (surface zinc fingers, sZFs). We show that sZFs enable sequence-specific labeling of living cells by dsDNA, and we develop a sequential labeling approach to image more than three cell types in mixed populations using three fluorophores. We demonstrate the versatility of sZFs through applications in which they serve as surrogate reporters, function as selective cell capture reagents and facilitate targeted cellular delivery of viruses

Evaluación de Parámetros de control obligatorio del agua potable proveniente del manantial Cuyuraya de la provincia de Huancané – Región Puno, 2019

El objetivo de este estudio fue el de evaluar los parámetros de control obligatorio de agua potable en la provincia de Huancané. Los puntos de monitoreo fueron seleccionados según la ubicación establecida por la Empresa Prestadora de Servicios “EPS NOR PUNO S.A.”; con los siguientes puntos: fuente principal de abastecimiento de agua (manantial Cuyuraya) y la red principal de abastecimiento (desde la primera vivienda, zona media y vivienda final) siendo un total de 6 puntos de monitoreo. Los parámetros evaluados según Reglamento de la Calidad del Agua para Consumo Humano DS N° 031 – 2010 – SA, fueron: pH, color, cloro residual, turbiedad, coliformes totales y termotolerantes, los cuales fueron analizados in-situ o en un laboratorio respectivamente, con un resultado de que solo 3 de los 6 parámetros evaluados cumplen con lo que indica el reglamento, debido a que Cloro Residual no llega al mínimo establecido de 0.5 mg/L, Coliformes Totales y Termotolerantes exceden en 16 UFC como máximo y <1.1 como mínimo, concluyendo que el agua que se distribuye en todo Huancané es directamente afectada por los niveles bajos de cloro residual, ocasionando que exista la presencia de coliformes (totales y termotolerantes), lo que afecta directamente a la calidad del agua que se distribuye en la provincia de Huancané.Trabajo de investigaciónJULIACAEscuela Profesional de Ingeniería AmbientalBiodiversidad y calidad ambienta

Variační rovnice na Möbiově pásce

In this paper, systems of second-order ordinary differential equations (or dynamical forms in Lagrangian mechanics), induced by the canonical embedding of the two-dimensional Möbius strip into the Euclidean space, are considered in the class of variational equations. For a given non-variational system, the conditions assuring variationality (Helmholtz conditions) for the induced system on the Möbius strip are formulated. The theory contributes to variational foundations of geometric mechanics.V tomto clánku je studována variacnost systému obycejných diferenciálních rovnic (dynamických forem v geometrické mechanice) druhého rádu, kterou indukuje kanonické vložení dvojrozmerné Möbiovy pásky do Euklidova prostoru. Pro daný nevariacní systém rovnic jsou formulovány nutné a postacující podmínky variacnosti (Helmholtzovy podmínky). Práce je príspevkem k variacním základum geometrické mechaniky na Möbiove pásce

Fluorescent and magnetic strategies facilitate sequence specific sorting of cells expressing surface LHEs

<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases"</p><p></p><p>Nucleic Acids Research 2007;35(8):2748-2758.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885675.</p><p>© 2007 The Author(s)</p> () Three populations of cells expressing different LHEs (I-AniI, I-AniI and H-DreI) were mixed at a 1:100:1 ratio and double stained with dsAni1-BT: SAv-PE and dsDre4-BT: SAv-Q655, followed by FACS. The resulting sorted populations were cultured for 5–7 days prior to analysis and subsequent rounds of sorting. In post-sort analyses, cells stained with dsAni1 and dsDre4 are shown in red and blue, respectively. () Enrichment of low frequency dsOligo-binding cells by MACS. IgM-negative DT40 cells expressing I-AniI (top row, third panel) were used as a background population into which IgM-positive B10 cells were added at a frequency of 0.1%. IgM-positive I-AniI cells were included at 0.5% to control for potential background dsOligo binding caused by surface immunoglobulin expression, leading to a total of 0.6% IgM-positive cells in the input population, the majority of which do not stain with dsAni1. This mixed population was stained and sorted using AutoMACS (see Methods section for details). The positive fraction was grown out and analyzed for IgM expression. Staining with dsAni1 confirmed that the enriched IgM-positive population primarily expressed wild-type I-AniI

Fluorescently conjugated dsOligos bind cell surface LHEs in a manner, which is sequence specific and easily resolved by flow cytometry

<p><b>Copyright information:</b></p><p>Taken from "Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases"</p><p></p><p>Nucleic Acids Research 2007;35(8):2748-2758.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885675.</p><p>© 2007 The Author(s)</p> () H-DreI is an engineered enzyme composed of domains derived from the I-CreI and I-DmoI LHEs. Its 23-bp recognition site (dsDre4, boxed) is therefore a complex of the natural target sequences bound by I-CreI (green) and I-DmoI (purple). The 19-bp I-AniI recognition site (dsAni1, boxed) was placed between stretches of five GC base pairs designed to enhance the formation and stability of the double-stranded complex. Single base-pair changes (dsDre4, dsDre4, dsAni1 and dsAni1) are indicated by red boxes and the cleavage sites by red arrows. The alternative I-AniI target sequence (dsAni2) containing two base-pair changes are shown in blue boxes. Conjugations with biotin at the 5′ termini are depicted, and Alexa Fluor 647 conjugated oligonucleotides for dsAni1 and dsAni1 were used in the flow cytometry cleavage assay. () Verification of efficient annealing of the complementary oligonucleotides run on a 3% agarose gel, with individual oligos (+S and −S) run as controls. () Flow cytometry analysis of clones stained with fluorescent dsOligos. Staining of I-AniI and H-DreI expressing clones in the presence of 10 mM Ca are shown, with shaded and open histograms representing SAv-PE-only controls and dsOligo-BT: SAv-PE stained cells, respectively. The dsOligos used for each stain are indicated in the upper right corner of the histograms