A Site‐Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9‐Mediated Genome Editing Strategies in CHO Cells

Targeted gene knockout and site‐specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (s ite‐s pecific i ntegration and g en ome al teration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR‐associated protein 9)‐mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual‐fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology.     

A QTL on the Ca7 chromosome of chickpea affects resistance to the root-lesion nematode Pratylenchus thornei

Molecular Breeding - Plant height is vital for crop yield by influencing plant architecture and resistance to lodging. Although lots of quantitative trait loci (QTLs) controlling plant height had...     

Synthesis of Novel Isocytosine Pseudonucleotide Analogues

Abstract

Ethyl dialkylphosphonoacetates were prepared from the corresponding dimethylalkylphosphites via the Arbuzov reaction with ethyl bromoacetate. The phosphonoacetates so produced were converted into enaminoacetates by reaction with DMF dimethylacetal and these were used as bidentate electrophiles for the synthesis of phosphonopyrimidones. Several of these compounds were tested for biological activity but none were found to possess antiviral activity.     

N-Substituted Acyclopurinenucleosides with Antiviral Activity

Abstract

N-Substituted Glyceropurines have been prepared. The N-dimethylaminomethyleneated and N-acetylated glyceroguanine derivatives have significant activity against Herpesviruses.     

Preparation,Characterization and Reactivity of Diastereomeric Sulfoxides of 8,2′-S-Anhydroadenosine

Abstract

The oxidation of 8,2′-S-anhydroadenosine (1a) has been investigated. The major product from the oxidation of 1a using 1-chlorobenzotriazole was the R-sulfoxide. The oxidation of 3′,5′-di-O-acetyl-8,2′-S-anhydroadenosine (1b) gave predominately the S-sulfoxide. These sulfoxides were found to be very succeptible to nucleophilic attack at C-8.     

Synthesis of Purine and Pyrimidine Trihydroxyacyclonucleosides

Abstract

The [[3-hydroxy-2-bis(hydroxymethyl)-l-propoxy]methyl] derivatives of adenine, guanine, cytosine and thymine have been synthesized and tested against herpesviruses.     

Using Manifold Learning for Atlas Selection in Multi-Atlas Segmentation

Multi-atlas segmentation has been widely used to segment various anatomical structures. The success of this technique partly relies on the selection of atlases that are best mapped to a new target image after registration. Recently, manifold learning has been proposed as a method for atlas selection. Each manifold learning technique seeks to optimize a unique objective function. Therefore, different techniques produce different embeddings even when applied to the same data set. Previous studies used a single technique in their method and gave no reason for the choice of the manifold learning technique employed nor the theoretical grounds for the choice of the manifold parameters. In this study, we compare side-by-side the results given by 3 manifold learning techniques (Isomap, Laplacian Eigenmaps and Locally Linear Embedding) on the same data set. We assess the ability of those 3 different techniques to select the best atlases to combine in the framework of multi-atlas segmentation. First, a leave-one-out experiment is used to optimize our method on a set of 110 manually segmented atlases of hippocampi and find the manifold learning technique and associated manifold parameters that give the best segmentation accuracy. Then, the optimal parameters are used to automatically segment 30 subjects from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). For our dataset, the selection of atlases with Locally Linear Embedding gives the best results. Our findings show that selection of atlases with manifold learning leads to segmentation accuracy close to or significantly higher than the state-of-the-art method and that accuracy can be increased by fine tuning the manifold learning process.     

Growth factor transgenes interactively regulate articular chondrocytes

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin‐like growth factor I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), transforming growth factor beta1 (TGF‐β1), bone morphogenetic protein‐2 (BMP‐2), and bone morphogenetic protien‐7 (BMP‐7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF‐I and FGF‐2 maximized cell proliferation. The three‐transgene group encoding IGF‐I, BMP‐2, and BMP‐7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell‐based articular cartilage repair. J. Cell. Biochem. 114: 908–919, 2013. © 2012 Wiley Periodicals, Inc.