Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells.

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng.

Selection of CHO host cell subclones with increased specific antibody production rates by repeated cycles of transient transfection and cell sorting.

Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by "hard fact" gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO-K1 and CHO-S host cells that possess an improved cellular machinery for transient antibody production.

Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases.

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem.

The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technology.

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming.

Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology.

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome.

A study on the temperature dependency and time course of the cold capture antibody secretion assay.

The cold capture assay as described by Brezinsky et al. [Brezinsky, S.C.

Resistance mapping and mode of action of a novel class of antibacterial anthranilic acids: evidence for disruption of cell wall biosynthesis.

Objectives: The aim of this study was to characterize the mechanism of action of a novel class of bacterial protein synthesis inhibitors identified in a high-throughput coupled transcription-translation assay.

Methods: Evaluation of the cross-resistance to antibiotics with known mechanisms of action, resistance mapping and biochemical characterization of a novel class of antibacterial anthranilic acids was performed.

Results: No cross-resistance to established classes of antibiotics was found.

Discovery and characterization of QPT-1, the progenitor of a new class of bacterial topoisomerase inhibitors.

QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (-)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization.

Elucidation of essential and nonessential genes in the Haemophilus influenzae Rd cell wall biosynthetic pathway by targeted gene disruption.

Targeted gene disruption by in vitro transposon mutagenesis has been used to identify the genes required for biosynthesis of the Haemophilus influenzae Rd cell wall under standard cultivation conditions. Of the 28 genes known to be associated with the cell wall biosynthetic pathway, 14 were determined to be essential.

Identification of the Haemophilus influenzae tolC gene by susceptibility profiles of insertionally inactivated efflux pump mutants.

Isogenic strains containing insertional disruptions of 10 Haemophilus influenzae Rd genes were investigated for their effects on the susceptibility of the organism to various classes of antimicrobial compounds. MIC results show that HI1462, which encodes an Escherichia coli TolC homolog, is the third component of the H. influenzae AcrAB pump.

Hierarchical linear models for the development of growth curves: an example with body mass index in overweight/obese adults.

When data are available on multiple individuals measured at multiple time points that may vary in number or inter-measurement interval, hierarchical linear models (HLM) may be an ideal option. The present paper offers an applied tutorial on the use of HLM for developing growth curves depicting natural changes over time. We illustrate these methods with an example of body mass index (BMI; kg/m(2)) among overweight and obese adults.

Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics.

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome.

Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.

Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S.