CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate

Dynamics of Trading Volume, Price, and Duration of Contractual Relationship

Abstract Agency theorists consider a firm as a nexus of contractual relationships. Contracting parties such as shareholders, debt holders, managers, input suppliers, advertising agencies and retailers may have information superior to outsiders concerning the firm's quality. In this paper, we show that the duration of contractual relationship within a firm has important implications on the dynamics of the price and trading volume of the firm's stock. If the duration of information asymmetry corresponds to that of contractual relationship, insiders with long-term relationship is shown to marginally prefer less aggressive trading strategies than those who can only access superior information temporarily. Such behavior makes it more difficult for the market maker * Address for correspondence: Ying-Ju Chen, phone: 212-998-0489, e-mail: [email protected], address: 44 W 4th Street, KMC 8-151, New York, NY 10012 1 to infer the existence of informed traders. The market maker will henceforth provide narrower bid-ask spreads, which attract discretionary liquidity traders to gather their trades in that period. With the long-term contractual relationship, uncertainty is resolved more slowly because the insider trades on her information advantage gradually. The long-term relationship also enlarges the liquidity traders' loss and discourages them from holding the firm's stock, and therefore reduces the amount of proceeds that the entrepreneur gathers when she makes financing. On the other hand, with the long-term relationship, a more efficient effort level can be expected, and thus the outcome of the investment plan will be higher. We conclude that the entrepreneur's profit as well as a firm's fundamental value will be influenced by the duration of the firm's internal contract. The optimal contractual duration depends on the adverse selection cost and the investment efficiency

Splint therapy for disc displacement with reduction of the temporomandibular joint. Part I: Modified mandibular splint therapy

AbstractThe aims of this preliminary study were to present a modified mandibular splint together with a treatment regimen and to evaluate their effects on the treatment of reciprocal joint sounds of the temporomandibular joint (TMJ). The study participants were recruited from 312 consecutive patients in the temporomandibular disorder clinic of a medical center in Taiwan from January 2003 to December 2003. From among these, 59 cases with typical reciprocal clicking were selected for this study. All participants were treated with a modified mandibular splint and then followed up for 6 months. Successful treatment was defined as leading to the disappearance of the joint sounds of TMJ, as described by patients. Based on clinical evaluation, the overall success rate was 71.2% (42/59) with minimal temporary complications. Patients with clicking at less than 3.5cm of interincisal opening had a success rate of 92.5%, which was higher than the success rate of patients with clicking at a mouth opening of 3.5cm or more. This study showed that a modified mandibular splint can be used to treat reciprocal clicking of the TMJ effectively and encouraged us to conduct further study on the efficacy of this splint to treat disc displacement with reduction of TMJ using magnetic resonance imaging examination

Development of a high-density CHO-C system enables rapid protein production in 10 days

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High-yield antibody production using targeted integration and engineering CHO host

To identify the high expression sites in the CHO cells, we employed NGS to analyze the integration sites of a high producing cell line (titer \u3e 3g/L). The pair-end reads with one read mapped to the vector and the other read mapped to the CHO reference genome are extracted to identify the integration sites. To test the expression activity of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed 4 integration sites are in the high producing cell line. Among the 4 integration site, one integration site was tested by CRISPR/Cas9 for target integration of antibody gene for expression. The target integrated cell pool present higher expression level (130 mg/L/copy) and less copy number when compared other integration sites. Through single-copy integration method, we can also achieve 60-150 mg/L/copy in a batch culture. About 80% of the single-copy cell clones were stable at generation 60. We have also applied the CHO-specific microarray transcriptomics technology to identify genes that contribute to high productivity. Transfection of our proprietary dual promoter vector J 1.0 resulting in 1.65 to 2.4 fold increase in the expression in engineered CHO DXB11 host. Through fed-batch process development, 3 – 5 g/L mAb productivity can be achieved through targeted integration and engineered CHO host