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. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering.

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.G., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W., Thill, G., 2003. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141-155] stands out as the most simple of single cell secretion assays which can be used to sort for high productivity in recombinant cell lines. At low temperatures the process of protein release from transport vesicles is assumed to be delayed as both vesicle fusion and product release is slowed, so that secreted proteins can be stained on the cell surface using a fluorescent antibody. Typically, the fluorescent signal obtained correlates to the cell specific production rate of the analysed cell. In the present study we compared staining of human antibody producing CHO cells performed at different temperatures and we observed the fluorescent signal over 24h. We found that the staining temperature did not influence signal intensity. The fluorescent signal was stable for 24h at 4 degrees C, decreased to 80% at room temperature (21 degrees C), while it decreased significantly already after 2h at 37 degrees C. Initially, the fluorescent signal was observed on the cell surface, however, at later stages it was found in compartments in the cytoplasm. Finally we compared differences in signal stability depending on whether the antibody used for staining bound to the light or heavy chain of the product and on whether the fluorescent label was a relatively stable protein (phycoerythrin) or a pH-dependent small molecule (FITC). Our results indicate that the secreted product is trapped by the staining antibody on the cell surface at all temperatures. Subsequently these aggregates are endocytosed by the cells, a process which is slowed down at low temperatures.

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. Biochemical and genetic characterization showed that the QPT-1 targets the beta subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

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. Resistance was mapped to SA1575, an essential, integral membrane protein predicted to be involved in polysaccharide biosynthesis. Biochemical analysis demonstrated the inhibition of cell wall biosynthesis. CONCLUSIONS: This novel class of antibacterial anthranilic acids inhibits cell wall biosynthesis. Resistance mapped to SA1575, which may represent a novel target for antibacterial drug discovery.

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.

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. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.

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. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.